Use of CSA compounds to stimulate stem cells and hair growth

Abstract

To provide methods for regenerating tissue and / or preventing tissue loss by stimulating stem cells.SOLUTION: A method for regenerating tissue and / or preventing tissue atrophy, the method comprising: providing a treatment composition comprising one or more cationic steroid antimicrobial (CSA) compounds in a carrier; administering the treatment composition to a targeted tissue of a subject; and the treatment composition stimulating tissue regeneration and / or preventing tissue atrophy at the targeted tissue to which it is applied.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Tissue damage occurs due to a combination of several factors, including injury, disease, genetic conditions or predisposition, infection, inflammation, stress, or autoimmune responses. The body utilizes stem cells to grow and maintain tissues. However, relying solely on the self-activation of endogenous stem cells cannot always prevent or reverse tissue deterioration. Damaged tissues cannot regenerate because tissue stem cells are not stimulated to promote the regeneration and maintenance of the associated tissue.

[0002] One type of tissue deterioration is hair loss. In animals that spend a lot of time outdoors, such as farm animals, hair loss makes exposed areas of skin more susceptible to sunburn, insect bites, and inflammation. In humans, thinning hair and baldness can cause psychological distress due to the impact on appearance.

[0003] When underlying infection or inflammation is associated with hair loss or other tissue damage, treatment for hair loss or tissue damage typically involves treating the underlying infection and allowing hair and tissue growth to resume naturally. Once the underlying condition is treated, hair and tissue growth often resumes, but it can take a long time for hair and tissue to return to their pre-infection thickness and health. When other causes of hair and tissue loss exist, treatment options may include medications (such as minoxidil (often sold under the brand names Rogaine® or Regaine®)), corticosteroid injections, hormone modification, immunosuppressants, hair transplant surgery, or cosmetics such as wigs.

[0004] While stem cell therapy has been known for some time, several limitations exist for its effective and targeted use in tissue regeneration. Stem cells are notoriously difficult to harvest and stimulate. Therefore, improved compositions and methods are needed to stimulate stem cells to promote tissue regeneration and / or prevent tissue degradation. Summary of the Invention [Means for solving the problem]

[0005] The present disclosure describes methods for regenerating tissue and / or preventing tissue atrophy or deterioration by administering one or more cationic steroid antimicrobial (CSA) compounds. In embodiments, the methods include: (1) providing a tissue regeneration composition (i.e., a therapeutic composition) comprising one or more CSA compounds and a carrier; (2) applying the tissue regeneration composition to a subject in need thereof; and (3) allowing the tissue regeneration composition to stimulate tissue regeneration in the subject.

[0006] The tissue regeneration properties of the compositions described herein are believed to be at least in part due to their ability to promote stem cell proliferation and migration, growth factor production by stem cells, or both. Stem cell proliferation may directly aid in tissue regeneration if the stimulated stem cells differentiate into the types of cells required to regenerate the targeted tissue. However, even if stem cells do not directly differentiate into the types of cells that constitute the regenerated tissue, they can themselves promote tissue regeneration by promoting the secretion of growth factors.

[0007] The tissue regeneration compositions can be administered by any suitable route of administration, including topical, oral, rectal, transdermal, inhalation, or injection, etc. In embodiments, the therapeutic compositions are formulated as creams, ointments, lotions, solutions, sprays, soaps, shampoos, or other preparations that are easily administered for topical application.

[0008] In a preferred embodiment, a tissue regeneration composition containing one or more CSA compounds is applied directly to a target tissue to stimulate local stem cells, thereby promoting tissue regeneration. Thus, the stem cells stimulated may be tissue (i.e., "adult") stem cells, such as mesenchymal stem cells (i.e., "mesenchymal cells"), or tissue-specific stem cells, such as follicular stem cells, hematopoietic stem cells, neural stem cells, or epithelial stem cells (e.g., present in the intestine or skin). Examples include stem cells present in bone marrow, peripheral blood, brain, spinal cord, dental pulp, blood vessels, skeletal muscle, epithelium of the skin and digestive system, cornea, retina, liver, pancreas, etc. Surprisingly, applying the tissue regeneration composition directly to the target tissue effectively stimulates tissue stem cells and induces corresponding tissue regeneration, without the need for separate stem cell harvesting and expansion.

[0009] In contrast, stem cells may be harvested separately and treated and / or mixed with a treatment composition containing one or more CSA compounds prior to application to the target tissue. Alternatively, a culture of stem cells may be treated with a treatment composition containing one or more CSA compounds, and then the conditioned media from the stem cell culture may be applied to the target tissue. In such embodiments, tissue stem cells (e.g., harvested from umbilical cord tissue, bone marrow, adipose tissue, and / or the tissue type of interest itself), induced pluripotent stem cells, and / or embryonic stem cells, as described above, may be utilized.

[0010] In one embodiment, the target tissue is hair-producing dermal tissue, and the tissue regeneration composition is administered to regenerate hair follicles, thereby stimulating hair growth. A method of stimulating hair growth may include the following steps: (1) providing a tissue regeneration composition comprising one or more CSA compounds and a carrier; (2) applying the tissue regeneration composition to a subject experiencing or at risk of hair loss; and (3) allowing the tissue regeneration composition to stimulate hair growth and / or prevent hair loss in the subject.

[0011] In embodiments, the tissue regeneration composition is applied to an anatomical subject infected with a microbial infection, such as a subject with a fungal and / or bacterial infection. In such embodiments, the antimicrobial activity of the composition can further promote tissue regeneration at the site of infection by removing the underlying microbial load that may contribute to tissue damage and / or prevent desired tissue growth. However, even in the absence of an antimicrobial effect, the growth-stimulating effect of the disclosed therapeutic methods has been found to function independently of the antimicrobial effect of the therapeutic method.

[0012] In embodiments, the tissue regeneration compositions are applied to treat hair loss associated with fungal or other microbial infections. In such embodiments, the antimicrobial activity of the compositions may aid in hair growth by eliminating the underlying infection causing hair loss. However, it has been found that the hair growth-promoting effects of the disclosed therapeutic agents act in addition to, and independently of, the antimicrobial effects of the therapeutic agents.

[0013] In a currently preferred embodiment, therapeutic compositions used to stimulate tissue regeneration and / or prevent tissue atrophy include CSA with a hydrolyzable (e.g., ester) bond. CSA compounds of this type generally have low production costs. Furthermore, such CSA compounds provide the desired activity when applied or administered, but are then naturally hydrolyzed and degraded to an inactive form, thereby minimizing concerns about long-term and / or environmental exposure.

[0014] Any CSA compound described herein, or any combination of such CSA compounds, can be utilized in a therapeutic composition. In a preferred embodiment, the therapeutic composition includes one or more CSA compounds having a hydrolyzable bond. Exemplary CSA compounds include CSA-44, CSA-142, CSA-144, CSA-145, CSA-146, and CSA-148, particularly CSA-44, CSA-142, CSA-144, and CSA-148. Alternatively, CSA-13 has demonstrated efficacy but does not contain a hydrolyzable bond. Compounds such as CSA-131, CSA-192, CSA-255, and CSA-256 are also expected to stimulate tissue regeneration but do not contain readily hydrolyzable linkages. CSA-13 and CSA-131 are more stable than CSA-192, CSA-255, and CSA-256, which contain hydrolyzable bonds. [Brief explanation of the drawings]

[0015] To illustrate the various features and concepts of the present disclosure, particular subject matter will be described more particularly by reference to specific embodiments that are illustrated in the accompanying drawings, with the understanding that these figures depict exemplary embodiments only and are not limiting in scope, and various embodiments will be described with additional specificity and detail using the accompanying drawings below.

[0016] [Figure 1A] FIG. 1A shows examples of cationic steroidal antibacterial compounds with ester or amide bonds at one or more of the R3, R7, and R12 positions. [Figure 1B] FIG. 1B shows examples of cationic steroid antibacterial compounds with ether linkages at one or more of the R3, R7, and R12 positions. [Figure 1C] FIG. 1C shows examples of cationic steroidal antibacterial compounds with an amide bond in the R18 group and ether or urethane bonds at the R3, R7, and R12 positions. [Figure 2A]Figures 2A-2C are photographs showing hair growth in mice in the control group and the two CSA-treated groups on days 7, 14, and 21 after depilation, respectively. [Figure 2B] Figures 2A-2C are photographs showing hair growth in mice in the control group and the two CSA-treated groups on days 7, 14, and 21 after depilation, respectively. [Figure 2C] Figures 2A-2C are photographs showing hair growth in mice in the control group and the two CSA-treated groups on days 7, 14, and 21 after depilation, respectively. [Figure 3A] Figures 3A and 3B are photographs of a horse suffering from hair loss before and after treatment with a CSA-based therapeutic composition. [Figure 3B] Figures 3A and 3B are photographs of a horse suffering from hair loss before and after treatment with a CSA-based therapeutic composition. DETAILED DESCRIPTION OF THE INVENTION

[0017] The embodiments disclosed herein will now be described with reference to more detailed embodiments, occasionally with reference to the accompanying drawings where appropriate. However, these embodiments may be embodied in different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the embodiments to those skilled in the art.

[0018] I. Overview of CSA Compounds Cationic steroid antimicrobial (CSA) compounds (also known as "CSA compounds," "CSA," CSA molecules, or "ceragenin" compounds) are synthetically produced small chemical compounds containing a sterol backbone with various charged groups (e.g., amine and cationic groups) attached. The amine and guanidine groups can be used to orientate the faces of the sterol backbone. CSAs are cationic and amphiphilic based on the functional groups attached to the backbone. CSAs are amphiphilic, with a hydrophobic face and a polycationic face.

[0019] Without wishing to be bound by any particular theory, it is theorized that the CSA compounds described herein act as antimicrobial agents (e.g., antibacterial, antifungal, and / or antiviral agents) by binding to and inserting into the cell membrane of microorganisms, such as bacteria, creating pores that allow leakage of ions and cytoplasmic materials important to the survival of the microorganism, thereby killing the affected microorganism.

[0020] Surprisingly, the use of CSA compounds has also been found to stimulate stem cells, thereby promoting tissue regeneration, independent of the compounds' antibacterial properties. A specific application of stimulating stem cells to regenerate tissue is the treatment of hair loss. Even when hair loss is associated with an underlying infection, administering one or more CSA compounds has been found to promote hair follicle regeneration, independent of treatment of the underlying infection. For example, hair regrowth has been found to be faster and thicker when using CSA compounds compared to the use of conventional topical antibacterial compounds to treat skin infections.

[0021] An example of a CSA compound is shown below as Formula I. As described in more detail below, the R group in Formula I can have a variety of different functionalities, which can result in specific and different properties being imparted to a given ceragenin compound. Furthermore, as one skilled in the art will appreciate, the sterol backbone can be formed of five- and / or six-membered rings, and therefore p, q, m, and n can independently be 1 (providing a six-membered ring) or 0 (providing a five-membered ring). Typically, the A, B, and C rings are six-membered rings, and the D ring is a five-membered ring. [ka]

[0022] The CSA compound can have the structure of Formula II, Formula III, or Formula IV: [ka]

[0023] The R groups are defined as follows: Formula II is a subset of Formula I where rings A, B, C, and D are six-membered rings; Formula III is a subset of Formula I where rings A, B, and C are six-membered rings and D is a five-membered ring; Formula IV is a subset of Formula I where the stereochemistry is defined and R3, R7, R 12 , and R 18 is a subset of Formula III where the R groups other than are defined as either hydrogen or methyl.

[0024] Numerous examples of CSA compounds of Formula I, Formula II, Formula III, and Formula IV that can be used to stimulate stem cells are shown in Figures 1A-1C.

[0025] Typically, CSAs as used herein are of two types: (1) CSAs having a cationic group attached to the sterol backbone by a hydrolyzable bond, and (2) CSAs having a cationic group attached to the sterol backbone by a non-hydrolyzable bond. For example, one type of hydrolyzable bond is an ester bond, and one type of non-hydrolyzable bond is an ether bond. The first type of CSA is "inactivated" by hydrolysis of the linkage connecting the cationic group to the sterol backbone, while the second type of CSA is more resistant to degradation and inactivation.

[0026] Numerous examples of CSA compounds that can be used in the embodiments described herein are shown in Figures 1A-1C. Non-limiting examples of CSAs having hydrolyzable linkages are listed in Figure 1A and include CSA-27, CSA-28, CSA-30, CSA-31, CSA-32, CSA-33, CSA-34, CSA-35, CSA-36, CSA-37, CSA-41, CSA-42, CSA-43, CSA-44, CSA-45, CSA-47, CSA-49, CSA-50, CSA-51, CSA-52, CSA-56, CSA-61, CSA-141, CSA-142, CSA-144, CSA-145, CSA-146, and CSA-148.

[0027] Non-limiting examples of CSAs with non-hydrolyzable bonds are shown in FIG. 1B and include CSA-13, CSA-90, CSA-131, CSA-136, CSA-137, and CSA-138.

[0028] Non-limiting examples of CSAs having both hydrolyzable and non-hydrolyzable links are shown in FIG. 1C and include CSA-190, CSA-191, CSA-192, CSA-255, CSA-256, and CSA-257.

[0029] In a currently preferred embodiment, the therapeutic composition used to stimulate hair growth and / or prevent hair loss is a CSA having a hydrolyzable (e.g., ester) bond. This type of CSA compound generally has low production costs. Furthermore, such CSA compounds provide the desired activity when applied or administered, but then naturally undergo hydrolysis and decomposition into an inactive form, thereby minimizing concerns about long-term exposure and / or environmental exposure.

[0030] In Formula I, Formula II, Formula III, and Formula IV, R3, R7, or R 12 At least two of may independently contain a cationic moiety attached to the sterol backbone via a hydrolyzable (e.g., ester) or non-hydrolyzable (e.g., ether) bond. The tail moiety is typically 18 is attached to Formula I. The tail moiety can be, for example, charged, uncharged, polar, non-polar, hydrophobic, or amphipathic, and can be selected to thereby tailor and / or provide desired properties of the CSA.

[0031] The activity of the CSA compound may be affected by the orientation of the substituents attached to the backbone structure. In one embodiment, the substituents attached to the backbone structure are oriented on a single face of the CSA compound. Thus, R3, R7, and R 12 may be located on a single face of Formula I, Formula II, Formula III, and Formula IV. 18may also be arranged on the same single surface.

[0032] II. Stem cell activation by CSA compounds

[0003] Embodiments described herein are directed to methods for regenerating tissue and / or preventing tissue atrophy or deterioration in a subject. In embodiments, the methods include: (1) providing a tissue regeneration composition (i.e., a therapeutic composition) comprising one or more CSA compounds and a carrier; (2) applying the tissue regeneration composition to a subject in need thereof; and (3) allowing the tissue regeneration composition to stimulate tissue regeneration in the subject.

[0033] In one embodiment, the target tissue is hair-producing dermal tissue, and the tissue regeneration composition is administered to regenerate hair follicles, thereby stimulating hair growth. In an embodiment, the method includes the steps of: (1) providing a tissue regeneration composition comprising one or more CSA compounds and a carrier, (2) applying the tissue regeneration composition to a subject in need thereof, and (3) allowing the tissue regeneration composition to stimulate hair growth and / or prevent hair loss in the subject.

[0034] Other potential therapeutic applications include regenerating tissue damaged as a result of, for example, cancer treatment (e.g., non-Hodgkin's lymphoma, leukemia), stroke, autoimmune diseases such as osteoarthritis and rheumatoid arthritis, spinal cord injury, brain injury, heart injury or damage, and type 1 diabetes.

[0035] In a preferred embodiment, tissue regeneration compositions containing one or more CSA compounds are applied directly to target tissues to stimulate local stem cells, thereby promoting tissue regeneration. Thus, the stem cells stimulated may be tissue (i.e., "adult") stem cells, such as mesenchymal stem cells (i.e., "stromal cells"), or tissue-specific stem cells, such as follicular stem cells, hematopoietic stem cells, neural stem cells, and epithelial stem cells (e.g., found in the intestine and skin). Examples include stem cells found in bone marrow, peripheral blood, brain, spinal cord, dental pulp, blood vessels, skeletal muscle, epithelium of the skin and digestive system, cornea, retina, liver, and pancreas. Surprisingly, applying tissue regeneration compositions directly to target tissues effectively stimulates tissue stem cells and induces corresponding tissue regeneration, without the need for separate stem cell harvesting and expansion.

[0036] In contrast, stem cells may be harvested separately and treated and / or mixed with a treatment composition containing one or more CSA compounds prior to application to the target tissue. Alternatively, a culture of stem cells may be treated with a treatment composition containing one or more CSA compounds, and then the conditioned media from the stem cell culture may be applied to the target tissue. In such embodiments, tissue stem cells (e.g., harvested from umbilical cord tissue, bone marrow, adipose tissue, and / or the tissue type of interest itself), induced pluripotent stem cells, and / or embryonic stem cells, as described above, may be utilized.

[0037] While embodiments that include harvesting stem cells are within the general scope of the present disclosure, they are less preferred than methods in which the therapeutic composition is applied directly to the target tissue because they require the additional steps of harvesting or harvesting the stem cells, mixing or activating the harvested stem cells, and optionally culturing the stem cells to harvest the conditioned medium.

[0038] The tissue regenerative properties of the compositions described herein are believed to be at least in part due to their ability to promote stem cell proliferation and migration, growth factor production by stem cells, or both. Stem cell proliferation can directly aid in tissue regeneration, such as when stimulated stem cells differentiate into the types of cells required for regeneration of the targeted tissue. However, even when stem cells do not directly differentiate into the types of cells that constitute the regenerated tissue, they can themselves promote tissue regeneration by stimulating the secretion of growth factors.

[0039] Without being bound by any particular theory, it is believed that CSA compounds can modulate one or more cellular receptors, such as formyl peptide receptor-like 1 (FPRL1) and / or other G protein-coupled receptors, which may induce various signaling pathways, such as enhanced expression of early growth response 1 (EGR1) and enhanced activation of mitogen-activated protein kinases (MAPKs), resulting in stem cell-stimulating effects, such as enhanced proliferation and migration of stem cells, enhanced production of extracellular growth factors, enhanced paracrine and / or endocrine signaling, or a combination thereof.

[0040] The tissue regeneration compositions can be administered by any suitable route of administration, including topical, oral, rectal, transdermal, inhalation, or injection, etc. In embodiments, the therapeutic compositions are formulated as creams, ointments, lotions, solutions, sprays, soaps, shampoos, or other preparations that are easily administered for topical application.

[0041] In embodiments, the therapeutic composition is applied to treat tissues having a microbial infection, such as a fungal or bacterial infection. In such embodiments, the antimicrobial activity of the composition can indirectly promote tissue regeneration by reducing or eliminating the underlying infectious burden in the target tissue. However, it will be understood that there are also stem cell stimulatory and tissue regenerative effects that function independently of the antimicrobial effect of the therapeutic agent.

[0042] For example, treating infected tissue in a subject with conventional antibacterial agents may result in comparable clearance of the underlying infection, but may result in less tissue recovery and a lower ability to resist tissue atrophy compared to a CSA-based therapeutic composition. Thus, the disclosed treatments effectively stimulate stem cells and promote tissue regeneration without relying on additional antibacterial activity.

[0043] The subject to which the therapeutic composition is applied may be an animal having injured tissue, experiencing tissue loss or atrophy, or at risk of tissue loss or atrophy. The tissue may be at risk of atrophy or degeneration due to, for example, injury, infection, a genetic condition, or normal or premature aging.

[0044] In certain applications in which the therapeutic composition is directed to hair follicle tissue to stimulate hair growth, the subject may be any mammal experiencing or at risk of hair loss. Examples include pets, livestock, laboratory animals, zoo animals, and humans. The actual or potential hair loss may be associated with an infection (e.g., dermatophytosis or other fungal skin infection) or may be associated with, for example, a genetic condition, a hormonal imbalance, burns, sunburn, or other damage to the dermal tissue.

[0045] In embodiments, the CSA-based therapeutic composition is applied in a relatively short-term or temporary regimen until the target tissue is sufficiently regenerated or until the underlying cause of tissue atrophy is eliminated. For example, if the problem is associated with an underlying infection, the therapeutic composition can be applied until the underlying infection is cured. In such situations, application of the CSA-based therapeutic composition can beneficially promote faster tissue recovery and / or provide more effective regeneration (e.g., thicker / more hair growth in hair follicle / hair application) compared to conventionally treating the underlying infection and waiting or expecting the tissue to heal.

[0046] In other embodiments, the CSA-based therapeutic composition is applied in a more continuous manner. For example, the therapeutic composition may be applied prophylactically to reverse or prevent tissue atrophy or deterioration. In such situations, the therapeutic composition may be applied multiple times a day (e.g., morning and night), daily, weekly, or as frequently as appropriate to provide sufficient tissue regeneration and maintenance. One example of such use is to reverse and / or prevent hair loss when a subject is suffering from or at risk of hair loss.

[0047] For topical application, the therapeutic composition may be administered using a pharmaceutically acceptable carrier, such as a solvent, surfactant, skin-penetrating agent (e.g., ethanol, isopropyl alcohol, other alcohols, dimethyl sulfoxide), oil, emulsion, water, and / or combinations thereof. The composition may be provided in the form of, for example, a liniment, lotion, ointment, cream, powder, wash, or spray. The composition may also be incorporated into another topical product, such as a shampoo, conditioner, soap, or hair care product. In other embodiments, the therapeutic composition may additionally or alternatively be administered using another non-topical route, such as injection, ingestion, or inhalation.

[0048] In embodiments, one or more CSA compounds are present in the treatment composition at about 0.01%, 0.1%, 0.2%, 0.3%, 0.5%, 1%, 2%, 3%, 5%, 10%, 15%, 20%, 25%, or 30% by weight, or within a range defined by any two of the foregoing percentage values. Presently preferred ranges are from about 0.1% to about 5%, or from about 0.2% to about 3%, or from about 0.3% to about 2%. In embodiments, one or more CSA compounds are present at a concentration of about 1 μg / ml, 5 μg / ml, 10 μg / ml, 25 μg / ml, 50 μg / ml, 100 μg / ml, 150 μg / ml, or 200 μg / ml, or within a range defined by any two of the foregoing concentration values.

[0049] In the foregoing examples, it will be understood that the upper concentration endpoint does not necessarily indicate that CSA concentrations above the upper endpoint are ineffective. Rather, the upper concentration endpoint defines a range within which effective activity is obtained without the need for additional CSA compound, thereby allowing for efficient use of CSA compounds in light of associated formulation costs. In some embodiments, such as when cost is less important than high activity, one or more CSA compounds may be included at concentrations higher than the aforementioned ranges.

[0050] In embodiments, treatment of a subject with a CSA-based therapeutic composition can halt or at least slow tissue degeneration, hi embodiments, treatment of a subject with a CSA-based therapeutic composition can stimulate tissue regeneration.

[0051] In embodiments, treatment of a subject with a CSA-based therapeutic composition can stop or at least slow hair loss. In embodiments, treatment of a subject with a CSA-based therapeutic composition can stimulate hair growth regrowth. Treatment of a subject with a CSA-based therapeutic composition can promote hair growth at a rate of about 1.2 to 5 times, or about 1.5 to 3 times, the hair regrowth rate observed with conventional treatment or no treatment.

[0052] Any CSA compound described herein, or any combination of such CSA compounds, can be utilized in a therapeutic composition. In a preferred embodiment, the therapeutic composition includes one or more CSA compounds having a hydrolyzable bond. Exemplary CSA compounds include CSA-44, CSA-142, CSA-144, CSA-145, CSA-146, and CSA-148, particularly CSA-44, CSA-142, CSA-144, and CSA-148. [Example]

[0053] III. Case studies Example 1 A mouse model was used to test the efficacy of a CSA-based therapeutic composition in stimulating stem cells and regenerating hair growth. Eight-week-old male C57BL / 6 mice were housed under environmentally monitored conditions and fed a standard rodent diet. The dorsal surface of each mouse was shaved with animal clippers, followed by application of a hair removal cream and washing away any remaining hair, as described by Jung MK et al. (Life Sci. 2015 May 1;128:39-46). The mice were then allowed to rest for 24 hours.

[0054] On the day of the experiment, all animals were weighed and randomly assigned to different treatment groups (n = 8) according to their weight. Randomized animals were identified by individual animal markings, and cage cards were used to identify groups.

[0055] Control mice, group 1, were treated topically with a cream-based vehicle. Group 2 animals received 100 mg of 0.5% CSA-44 topically once daily for three weeks. Group 3 animals received 100 mg of the test article in 2.0% CSA-44 topically every other day for three weeks. Details of the test article dose levels and each treatment group are shown in Table 1. [Table 1]

[0056] To select an appropriate and tolerable dose for this efficacy study, a preliminary tolerability study was conducted using C57BL / 6 mice. Based on the results, 100 mg of a 0.5% CSA-44 formulation was applied evenly to the shaved area of ​​the dorsal skin once daily for 3 weeks. Similarly, 100 mg of a 2% CSA-44 formulation was administered topically every other day for 3 weeks. 100 mg of the cream vehicle was applied topically to the shaved area once daily for 3 weeks.

[0057] Hair growth scoring Hair growth scores were assessed using the following scoring pattern described by Vegesna et al. (Endocrinology 143(11):4389-4396). A score of "0" indicates no change in hair growth compared to the day of hair removal, and a score of "10" indicates complete hair growth on all areas of the dorsal skin. Darkening and hair growth were observed twice a week for three weeks. To confirm the onset of hair growth and the hair growth pattern, the back skin of the mice was photographed weekly with a digital camera. [Table 2]

[0058] Animal weights were recorded before randomization (pre-dose) and twice weekly throughout the study. All animals were observed once daily for clinical signs and twice daily (morning and evening) for mortality and morbidity. No mortality was observed during the study period. After the study period, animals were killed according to standard protocols using CO2-induced euthanasia.

[0059] result Hair growth scores and body weights were recorded twice weekly throughout the study. On day 21, the percent change in hair growth was calculated by comparing treated mice with a vehicle-treated control group.

[0060] Effect of CSA-44: Animals treated with 0.5% CSA-44 experienced a 72% increase in hair growth (p<0.05) compared to animals treated with vehicle. After three weeks of treatment, mice had an average hair growth score of 6.88, compared to a score of 4 for the control group.

[0061] Animals treated with the 2% CSA-44 cream experienced a significant increase in hair growth of 66% (p<0.05) compared to the vehicle group. After three weeks of treatment, animals achieved an average hair growth score of 6.63, compared to an average score of 4 for the vehicle control group. [Table 3]

[0062] In the CSA-44 group, the area of ​​skin with black hair was larger than in the control group. After two weeks, many new hairs were observed growing from the hair follicles on the depilated back skin, and the skin color had changed to dark gray. Some parts of the skin were still pink, but hair growth was evident in some areas. After three weeks, the area of ​​skin with black hair in the CSA-44 group was larger than that in the control group. Photographs showing the progression of hair growth are shown in Figures 2A-2C. Figure 2A shows hair growth on day 7 after removal, Figure 2B shows hair growth on day 14 after removal, and Figure 2C shows hair growth on day 21 after removal. [Example]

[0063] Example 2 A treatment composition containing CSA-44 was applied to a horse with "summer itch" symptoms associated with a fungal skin infection. Figure 2A is a photograph of the horse before treatment. As shown, the horse had several visible patches of hair loss. Figure 2B is a photograph of the horse 18 days after treatment. Figure 2B is a photograph of the horse 18 days after treatment, showing hair regrowth that completely replaced the previous patches and was thick and lush. Following treatment of such fungal infections, the typical time for hair regrowth in a horse is approximately 28 days. Treatment with CSA resulted in hair regrowth 1.56 times faster than conventional treatment. [Example]

[0064] Example 3 A CSA-based therapeutic composition comprising one or more of CSA-44, CSA-142, CSA-144, CSA-145, CSA-146, or CSA-148 is applied to a skin area infected with a dermatophyte of a first mammalian subject. A conventional antifungal composition comprising clotrimazole is applied to a skin area infected with a dermatophyte of a second mammalian subject. A conventional antifungal composition comprising tioconazole is applied to a dermal area infected with a dermatophyte of a third mammalian subject. A conventional antifungal composition comprising tolnaftate is applied to a skin area infected with a dermatophyte of a fourth mammalian subject. A conventional antifungal composition containing terbinafine is applied to a skin area infected with a dermatophyte of a fifth mammalian subject. A conventional antifungal composition containing miconazole is applied to a skin area infected with a dermatophyte of a sixth mammalian subject. A conventional antifungal composition containing nystatin is applied to a skin area infected with a dermatophyte of a seventh mammalian subject. A conventional antifungal composition containing butenafine is applied to the dermatophyte-infected skin site of an eighth mammalian subject. A conventional antifungal composition containing fluconazole is applied to the dermatophyte-infected skin site of a ninth mammal. A conventional antifungal composition containing terconazole is applied to the dermatophyte-infected skin area of ​​a tenth mammal.

[0065] All subjects were confirmed to have been cured of their dermatophyte infections. Hair growth was confirmed to recover approximately 1.5 to 5 times faster in the affected area of ​​the first subject than in the affected areas of subjects 2 to 10. [Example]

[0066] Example 4 A CSA composition containing one or more of CSA-44, CSA-142, CSA-144, CSA-145, CSA-146, or CSA-148 is applied daily to the scalp of a male human experiencing early-stage hair loss. Hair loss ceases approximately 1-10 days after initiation of treatment. Hair regrowth begins approximately 5-30 days after initiation of administration.

[0067] IV. Details of CSA Compounds Exemplary CSA compounds and methods for their preparation are disclosed in U.S. Patent Nos. 6,350,738, 6,486,148, 6,767,904, 7,598,234, 7,754,705, 8,691,252, 8,975,310, 9,434,759, 9,527,883, 9,943,614, 10,155, 788, 10,227,376, 10,370,403, and 10,626,139, U.S. Patent Publication Nos. 2016 / 0311850 and 2017 / 0210776, and U.S. Application Nos. 63 / 025,255 and 63 / 028,249, which are incorporated herein by reference. Those skilled in the art will recognize compounds within the general formulas described herein and understand their preparation in light of the references and examples cited herein.

[0068] The CSA compound can have the structure of Formula I, Formula II, Formula III, and / or Formula IV. Formula III is a compound represented by the formula: R 15 and the annular carbon to which it is attached. Formula IV differs from Formulas I and II by omitting the stereochemistry and R3, R7, R 12 , and R 18 Formula III is more particularly defined for all R groups other than [ka] .

[0069] In embodiments of Formulas I, II, III, and IV, R, R, and R 12 At least two of the linkers may independently comprise a cationic moiety (e.g., an amino group or a guanidino group) attached to the steroid backbone via a hydrolyzable or non-hydrolyzable linkage. In embodiments of the present disclosure, the linkage is preferably hydrolyzable, but is stable under sterilization and storage conditions and hydrolyzable under physiological conditions. Such cationic functional groups (e.g., amino groups or guanidino groups) may be separated from the backbone by at least one, two, three, four, or more atoms.

[0070] The tail is R 18 The tails may be attached to a sterol backbone with a carboxyl group, may have variable chain lengths or sizes, and may be charged or uncharged, polar or non-polar, hydrophobic, or amphipathic. The tails can be used to select the hydrophobicity / hydrophilicity of the ceragenin compound. CSA compounds with different degrees of hydrophobicity / hydrophilicity may have different uptake rates into different target microorganisms.

[0071] The "R" groups described herein may be substituted or unsubstituted, unless otherwise specified.

[0072] With respect to CSA compounds of formula I, II, and III (and with respect to formula IV, if not already specified): The fused rings A, B, C, and D may each independently be saturated or fully or partially unsaturated, provided that at least two of A, B, C, and D are saturated and the rings A, B, C, and D form a ring system. Other ring systems can also be used, including compounds with backbones having fused five-membered rings and / or combinations of five- and six-membered rings. R1~R 18is hydrogen, hydroxyl, alkyl, hydroxyalkyl, alkyloxyalkyl, alkylcarboxyalkyl, terpenylcarboxyalkyl, terpenylcarbonyloxyalkyl, terpenylamidoalkyl, terpenylaminoalkyl, terpenyloxyalkyl, alkylaminoalkyl, alkylaminoalkylamino, alkylaminoalkylamino, aminoalkyl, aryl, arylaminoalkyl, haloalkyl, alkenyl, alkynyl, oxo, a linking group attached to a second steroid, aminoalkylretanyl, aminoalkenylretanyl, aminoarylretanyl, aminoalkyloxo independently selected from the group consisting of hydroxy, aminoalkylcarboxy, aminoalkyloxyalkyl, aminoalkylaminocarbonyl, aminoalkylcarboxamide, di(alkyl)aminoalkyl, HN—HC(Q5)—(C═O)—O—, HN—HC(Q5)—(C═O)—NH—, azidoalkyloxy, cyanoalkyloxy, PG-HN—HC(Q5)—(C═O)—O—, guanidinoalkyloxy, quaternary ammonium alkylcarboxy, and guanidinoalkylcarboxy, where Q5 is the side chain of any amino acid (including the side chain of glycine, i.e., H), and PG is an amino protecting group; and R5, R8, R9, R 10 , R 13 , R 14 and R 17 When any of the rings A, B, C, and D is unsaturated, it is independently deleted to complete the valence of the carbon atom at that site, However, R 1-4 , R6, R7, R 11 , R 12 , R 15 , R 16 , R 17 and R 18are independently selected from the group consisting of aminoalkyl, aminoalkyloxy, aminoalkylcarboxyalkyl, alkylaminoalkyl, alkylaminoalkylamino, alkylaminoalkylamino, aminoalkylcarboxy, arylaminoalkyl, aminoalkyloxyamino, alkylaminocarbonyl, aminoalkylcarboxamido, di(alkyl)aminoalkyl, aminoalkyluretanyl, aminoalkenylurethaneyl, aminoalkynyluretanyl, aminoallyluretanyl, HN—HC(Q5)—C(O)—O—, HN—HC(Q5)—C(O)—N(H)—, azidoalkyloxy, cyanoalkyloxy, PG-HN—HC(Q5)—C(O)—O—, guanidinoalkyloxy, quaternary ammoniumalkylcarboxy, and guanidinoalkylcarboxy.

[0073] In an embodiment, R1 to R4, R 6、 R7, R 11、 R 12 ,R, 15 , R 16 , and R 18 is hydrogen, hydroxyl, substituted or unsubstituted (C1-C 22 ) alkyl, substituted or unsubstituted (C1-C 22 ) hydroxyalkyl, substituted or unsubstituted (C1-C 22 )Alkyloxy-(C1-C 22 ) alkyl, substituted or unsubstituted (C1-C 22 ) alkylcarboxy-(C1-C 22 ) alkyl, substituted or unsubstituted (C5-C 25 ) terpenylcarboxy-(C1-C 22 ) alkyl, substituted or unsubstituted (C5-C 25 ) terpenylcarbonyloxy-(C1-C 22 ) alkyl, substituted or unsubstituted (C5-C 25 ) terpenylcarboxamido-(C1-C 22 ) alkyl, substituted or unsubstituted (C5-C 25 ) terpenylamino-(C1-C 22) alkyl, (C5-C 25 ) Terpenyloxyio-(C1-C 22 ) alkyl, substituted or unsubstituted (C1-C 22 )Alkylamino-(C1-C 22 ) alkyl, substituted or unsubstituted (C1-C 22 )Alkylamino-(C1-C 22 ) alkylamino, substituted or unsubstituted (C1-C 22 )Alkylamino-(C1-C 22 ) alkylamino, substituted or unsubstituted (C1-C 22 )aminoalkyl, substituted or unsubstituted aryl, substituted or unsubstituted arylamino-(C1-C 22 ) alkyl, substituted or unsubstituted (C1-C 22 ) haloalkyl, substituted or unsubstituted (C2-C6) alkenyl, substituted or unsubstituted (C2-C6) alkynyl, oxo, a linking group attached to a second steroid, substituted or unsubstituted (C1-C 22 )aminoalkyl ethoxylate, substituted or unsubstituted (C2-C 22 ) aminoalkenyl ethoxylate, substituted or unsubstituted (C2-C 22 ) aminoalkynylretanyl, and substituted or unsubstituted aminoarylretanyl, substituted or unsubstituted (C-C 22 )aminoalkyloxy, or unsubstituted (C1-C 22 )aminoalkylcarboxy, substituted or unsubstituted (C1-C 22 )aminoalkyloxy-(C1-C 22 ) alkyl, substituted or unsubstituted (C1-C 22 )aminoalkylaminocarbonyl, substituted or unsubstituted (C1-C 22 ) aminoalkylcarboxamide, substituted or unsubstituted di(C1-C 22 )Alkylamino-(C1-C 22 ) alkyl, H2N-HC(Q5)-(C=O)-O-, H2N-HC(Q5)-(C=O)-NH-, substituted or unsubstituted (C1-C 22 ) azidoalkyloxy, substituted or unsubstituted (C1-C 22) cyanoalkyloxy, PG-HN-HC(Q5)-(C=O)-O-, substituted or unsubstituted (C1-C 22 ) guanidinoalkyloxy, substituted or unsubstituted quaternary ammonium (C1-C 22 ) alkylcarboxy, and substituted or unsubstituted (C-C 22 ) guanidinoalkylcarboxy, Q5 is the side chain of an amino acid (including the side chain of glycine, i.e., H), PG is an amino protecting group, and

[0074] R5, R8, R9, R 10 , R 13 , R 14 and R 17 is independently deleted if any of rings A, B, C, or D is unsaturated to complete the valence of the carbon atom at that site, or R5, R8, R9, R 10 , R 13 and R 14 is hydrogen, hydroxyl, (C1-C 22 ) alkyl, (C1-C 22 ) hydroxyalkyl, (C1-C 22 )Alkyloxy-(C1-C 22 ) alkyl, (C1-C 22 ) aminoalkyl, aryl, (C1-C 22 ) haloalkyl, (C2-C6) alkenyl, (C2-C6) alkynyl, oxo, a linking group attached to a second steroid, (C1-C 22 )aminoalkyloxy, (C1-C 22 ) aminoalkylcarboxy, (C1-C 22 )aminoalkylaminocarbonyl, di(C1-C 22 alkyl)amino-(C1-C 22 ) alkyl, H2N-HC(Q5)-C(O)-O-, H2N-HC(Q5)-C(O)-N(H)-, (C1-C 22 ) azidoalkyloxy, (C1-C 22 ) cyanoalkyloxy, PG-HN-HC(Q5)-C(O)-O-, (C1-C 22 ) guanidinoalkyloxy, and (C1-C22 ) guanidinoalkylcarboxy, Q5 is the side chain of an amino acid, and PG is an amino protecting group;

[0075] However, R 1-4 , R6, R7, R 11 , R 12 , R 15 , R 16 , R 17 , and R 18 At least two or three of the following are 22 )aminoalkyl, (C1-C 22 )aminoalkyloxy, (C1-C 22 ) alkylcarboxy-(C1-C 22 ) alkyl, (C1-C 22 )Alkylamino-(C1-C 22 ) alkylamino, (C1-C 22 ) alkylamino-(C1-1C 22 ) alkylamino, (C1-C 22 )aminoalkylcarboxy, arylamino-(C1-C 22 ) alkyl, (C1-C 22 )aminoalkyloxy, (C1-C 22 )aminoalkylaminocarbonyl, (C1-C 22 ) Aminoalkylcarboxamide, Quaternary ammonium (C1-C 22 ) alkyl carboxy, di(C1-C 22 alkyl)amino-(C1-C 22 ) alkyl, (C1-C 22 ) Aminoalkyl ethoxylates, (C2-C 22 ) aminoalkenyl ethoxylate, (C2-C 22 ) Aminoalkynylretanyl, aminoarylretanyl, H2N-HC(Q5)-C(O)-O-, H2N-HC(Q5)-C(O)-N(H)-, (C1-C 22 ) azidoalkyloxy, (C1-C 22 ) cyanoalkyloxy, PG-HN-HC(Q5)-C(O)-O-, (C1-C 22 ) guanidinoalkyloxy, and (C1-C 22) guanidinoalkylcarboxy.

[0076] In an embodiment, R1, R2, R4, R5, R6, R8, R9, R 10 , R 11 , R 13 , R 14 , R 15 , R 16 and R 17 is independently selected from the group consisting of hydrogen and unsubstituted (C1-C6) alkyl.

[0077] In an embodiment, R1, R2, R4, R5, R6, R8, R 10 , R 11 , R 14 , R 16 , and R 17 are hydrogen, and R and R 13 are each methyl.

[0078] In embodiments, R, R, R 12 and R 18 are independently hydrogen, (C1-C6) alkyl, (C1-C6) hydroxyalkyl, (C1-C 16 ) alkyloxy-(C1-C5) alkyl, (C1-C 16 ) alkylcarboxy-(C1-C5) alkyl, (C1-C 16 ) alkylamino-(C1-C5) alkyl, (C1-C 16 ) alkylamino-(C1-C5) alkylamino, (C1-C 16 )Alkylamino-(C1-C 16 ) alkylamino, (C5-C 25 ) terpenylcarboxy-(C1-C5) alkyl, (C5-C 25 ) terpenylcarbonyloxy-(C1-C5) alkyl, (C5-C 25 ) terpenylcarboxamido-(C1-C5) alkyl, (C5-C 25 ) terpenylamino-(C1-C5) alkyl, (C5-C 25) terpenyloxy-(C1-C5) alkyl, (C1-C6) aminoalkylretanyl, (C2-C6) aminoalkenylretanyl, (C2-C6) aminoalkynylretanyl, aminoarylretanyl, (C1-C 16 )aminoalkyl, arylamino-(C1-C5)alkyl, (C1-C5)aminoalkyloxy, (C1-C 16 )aminoalkyloxy-(C1-C5) alkyl, (C1-C5) aminoalkylcarboxy, (C1-C5) aminoalkylamino-carbonyl, (C1-C5) aminoalkylcarboxy-amide, di(C1-C5 alkyl)amino-(C1-C5) alkyl, (C1-C5) guanidinoalkyloxy, quaternary ammonium (C1-C 16 ) alkylcarboxy, and unsubstituted (C-C 16 ) guanidinoalkylcarboxy.

[0079] In an embodiment, R1, R2, R4, R5, R6, R8, R 10 , R 11 R 14 , R 16 , and R 17 are hydrogen, and R and R 13 are methyl, respectively.

[0080] In embodiments, R, R, R 12 and R 18 is independently selected from the group consisting of aminoalkyloxy, aminoalkylcarboxy, alkylaminoalkyl, alkoxycarbonylalkyl, alkylcarbonylalkyl, di(alkyl)aminoalkyl, alkylcarboxyalkyl, hydroxyalkyl, terpenylcarboxyalkyl, terpenylcarbonyloxyalkyl, terpenylcarboxamidoalkyl, terpenylaminoalkyl, terpenyloxyalkyl, aminoalkylretanyl, aminoalkenylretanyl, aminoalkynyluretanyl, aminoarylretanyl, and the like.

[0081] In embodiments, R3, R7, and R 12is independently selected from the group consisting of aminoalkyloxy, aminoalkylcarboxy, aminoalkyluretanyl, aminoalkenyluretanyl, aminoalkynyluretanyl, and aminoaryluretanyl.

[0082] In embodiments, R 18 is selected from the group consisting of alkylaminoalkyl, alkoxycarbonylalkyl, alkylcarbonyloxyalkyl, alkylcarbonylalkyl, di(alkyl)aminoalkyl, alkylcarboxyalkyl, hydroxyalkyl, terpenylcarboxyalkyl, terpenylcarbonyloxyalkyl, terpenylcarboxamidoalkyl, terpenylaminoalkyl, and terpenyloxyalkyl.

[0083] In embodiments, one or more of rings A, B, C, and D is a heterocycle.

[0084] In embodiments, rings A, B, C, and D are non-heterocyclic.

[0085] The compounds and compositions disclosed herein can be optionally prepared as salts, which advantageously result in cationization when one or more amine groups are protonated. As used herein, the term "salt" is a broad term and should be given its usual and customary meaning to those skilled in the art (it is not limited to any special or customized meaning), and refers without limitation to salts of compounds. In embodiments, the salts are acid addition salts of compounds. Salts can be obtained by reacting compounds with inorganic acids such as hydrohalic acids (e.g., hydrochloric acid or hydrobromic acid), sulfuric acid, nitric acid, phosphoric acid, and phosphonic acid. Furthermore, a salt can also be obtained by reacting the compound with an organic acid such as an aliphatic or aromatic carboxylic acid, sulfonic acid, or sulfinic acid, for example, formic acid, acetic acid, propionic acid, glycolic acid, pyruvic acid, malonic acid, maleic acid, fumaric acid, trifluoroacetic acid, benzoic acid, cinnamic acid, mandelic acid, succinic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, nicotinic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, stearic acid, muconic acid, butyric acid, phenylacetic acid, phenylbutyric acid, valproic acid, 1,2-ethanedisulfonic acid, 2-hydroxyethanedisulfonic acid, benzenesulfonic acid, 2-naphthalenesulfonic acid, or 1,5-naphthalenedisulfonic acid (NDSA). Salts can also be obtained by reacting a compound with a base to form alkali metal salts such as ammonium salts, lithium salts, sodium salts, potassium salts, and the like; alkaline earth metal salts such as calcium salts, magnesium salts, aluminum salts, and the like; salts of organic bases such as dicyclohexylamine; salts of organic bases such as N-methyl-D-glucamine, tris(hydroxymethyl)methylamine, C1-C7 alkylamines, cyclohexylamine, dicyclohexylamine, triethanolamine, ethylenediamine, ethanolamine, diethanolamine, triethanolamine, tromethamine, and the like; salts with amino acids such as arginine and lysine; or salts of inorganic bases such as aluminum hydroxide, calcium hydroxide, potassium hydroxide, sodium carbonate, sodium hydroxide, and the like.

[0086] In embodiments, the salt is a hydrochloride salt. In embodiments, the salt is a monohydrochloride, dihydrochloride, trihydrochloride, or tetrahydrochloride salt. Additional examples of salts include sulfate addition salts, sulfonic acid addition salts, disulfonic acid addition salts, 1,5-naphthalenedisulfonic acid addition salts, sulfate ester salts, and bisulfate ester salts.

[0087] Including but not limited to: R1, R2, R3, R4, R5, R6, R7, R8, R9, R 10 , R 11 , R 12 , R 13 , R 14 , R 15 , R 16 , R 17 , and R 18 "R" groups such as represent substituents that can be attached to the sterol backbone. Unless otherwise specified, R groups can be substituted or unsubstituted.

[0088] A "ring" can be heterocyclic or carbocyclic. "Saturated" means a ring in which each atom is hydrogen or substituted so that the valence of each atom is satisfied. "Unsaturated" means a ring in which the valence of each atom of the ring may not be satisfied with hydrogen or other substituents. For example, adjacent carbon atoms in a fused ring can be double-bonded to each other. Unsaturation can also occur when R5 and R9; R8 and R9; 10 ; and R 13 and R 14 It may also include deleting at least one of the following pairs and completing the valence of the carbon atom of the ring at the deleted position with a double bond, as in:

[0089] When a group is "substituted," it can be substituted with one, two, three, or more of the indicated substituents, which can be the same or different, each replacing a hydrogen atom. If no substituents are indicated, the indicated "substituted" group can be any of alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, acylalkyl, alkoxyalkyl, aminoalkyl, amino acid, aryl, heteroaryl, heteroalicyclyl, aralkyl, heteroaralkyl, (heteroalicyclyl)alkyl, hydroxy, protected hydroxyl, alkoxy, aryloxy, acyl, mercapto, alkylthio, arylthio, cyano, halogen (e.g., F, Cl, B r, I), thiocarbonyl, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, S-sulfonamido, N-sulfonamido, C-carboxy, protected C-carboxy, O-carboxy, isocyanato, thiocyanato, isothiocyanato, nitro, oxo, silyl, sulfenyl, sulfinyl, sulfonyl, haloalkyl, haloalkoxy, trihalomethanesulfonyl, trihalomethanesulfonamido, amino, monosubstituted amino group, disubstituted amino group, R a O(CH2) m O-, R b (CH2) n O-, R c C(O)O(CH2) p O- and protected derivatives thereof. Substituents may be attached to a group at more than one point of attachment. For example, an aryl group may be substituted at two points of attachment with heteroaryl groups to form a fused polycyclic aromatic ring system. Biphenyl and naphthalene are two examples of aryl groups substituted with a second aryl group. Groups not expressly designated as substituted or unsubstituted may be considered to be either substituted or unsubstituted.

[0090] "C" where "a" and "b" are integers a " or "C a ~C bThe term "a" refers to the number of carbon atoms in an alkyl, alkenyl, or alkynyl group, or the number of carbon atoms in the ring of a cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, or heteroalicyclyl group. That is, the alkyl, alkenyl, alkynyl, cycloalkyl ring, cycloalkenyl ring, cycloalkynyl ring, aryl ring, heteroaryl ring, or heteroalicyclyl ring can contain from "a" to "b" carbon atoms. For example, a "C1-C4 alkyl" group refers to all alkyl groups having 1 to 4 carbon atoms, i.e., CH3-, CH3CH2-, CH3CH2CH2-, (CH3)2CH-, CH3CH2CH2CH2-, CH3CH2CH(CH3)-, (CH3)2CHCH2-, and (CH3)3C-. When "a" and "b" are not specified for an alkyl, alkenyl, alkynyl, cycloalkylcycloalkenyl, cycloalkynyl, aryl, heteroaryl, or heteroalicyclyl group, the broadest range described by those definitions is intended.

[0091] "Alkyl" refers to a linear or branched hydrocarbon chain consisting of a fully saturated (no double or triple bonds) hydrocarbon group. The alkyl group may have 1 to 25 carbon atoms. (As used herein, a numerical range, such as "1 to 25," refers to each integer within the range. For example, "1 to 25 carbon atoms" means that the alkyl group may be composed of 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc. For example, "1 to 25 carbon atoms" means that alkyl groups consisting of 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., may be present, up to a total of 25 carbon atoms.) The alkyl group may also be a medium-sized alkyl having 1 to 15 carbon atoms. The alkyl group may also be a lower alkyl having 1 to 6 carbon atoms. The alkyl group of a compound may be designated as "C4" or "C1-C4 alkyl" or similar. By way of example only, "C1-C4 alkyl" indicates that the alkyl chain has 1 to 4 carbon atoms, i.e., the alkyl chain is selected from methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, and t-butyl. Representative alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl, pentyl, and hexyl. Alkyl groups can be substituted or unsubstituted.

[0092] "Alkenyl" refers to an alkyl group containing one or more double bonds in a linear or branched hydrocarbon chain. An alkenyl group may have 2 to 25 carbon atoms. (As used herein, numerical ranges such as "2 to 25" refer to each integer within the range. For example, "2 to 25 carbon atoms" means that the alkenyl group may be composed of 2, 3, or 4 carbon atoms. For example, "2 to 25 carbon atoms" refers to alkenyl groups containing 2, 3, 4, etc. carbon atoms, up to 25 carbon atoms; however, this definition also covers occurrences of the term "alkenyl" without a specified numerical range.) An alkenyl group may also be a medium-sized alkenyl having 2 to 15 carbon atoms. An alkenyl group may also be a lower alkenyl having 1 to 6 carbon atoms. The alkenyl group of a compound may be designated "C4" or "C2-C4 alkyl" or similar. An alkenyl group may be unsubstituted or substituted.

[0093] "Alkynyl" refers to an alkyl group having one or more triple bonds in its linear or branched hydrocarbon chain. An alkynyl group may have 2 to 25 carbon atoms. (As used herein, numerical ranges such as "2 to 25" refer to each integer in the specified range; for example, "2 to 25 carbon atoms" means that the alkynyl group may be composed of 2, 3, or 4 carbon atoms, etc. For example, "2 to 25 carbon atoms" refers to alkynyl groups composed of 2, 3, 4, etc. carbon atoms, up to 25 carbon atoms, although this definition also covers occurrences of the term "alkynyl" without a specified numerical range.) An alkynyl group may also be a medium-sized alkynyl having 2 to 15 carbon atoms. An alkynyl group may also be a lower alkynyl having 2 to 6 carbon atoms. The alkynyl group of a compound may be designated "C4" or "C2-C4 alkyl" or similar. An alkynyl group may be unsubstituted or substituted.

[0094] "Aryl" means a carbocyclic (all carbon) monocyclic or polycyclic aromatic ring system (including fused ring systems in which two carbocyclic rings share a chemical bond) that has a completely delocalized pi-electron system throughout all rings. Aryl groups can have a variety of carbon atoms. For example, aryl groups can be C6-C6 14 Aryl groups, C6-C 10 It can be an aryl group, or a C6 aryl group (provided that C6-C 10 The definition of aryl covers occurrences of "aryl" when no numerical range is specified.) Examples of aryl groups include, but are not limited to, benzene, naphthalene, azulene, etc. Aryl groups can be substituted or unsubstituted.

[0095] "Aralkyl" and "aryl(alkyl)" refer to an aryl group bonded as a substituent via a lower alkylene group. The aralkyl group may have 6 to 20 carbon atoms. (As used herein, numerical ranges such as "6 to 20" refer to each integer within the range; for example, "6 to 20 carbon atoms" means that the aralkyl group may be composed of 6 carbon atoms, 7 carbon atoms, 8 carbon atoms, etc. However, this definition also covers occurrences of the term "aralkyl" where no numerical range is specified.) The lower alkylene and aryl groups of an aralkyl may be substituted or unsubstituted. Examples include, but are not limited to, benzyl, 2-phenylalkyl, 3-phenylalkyl, and naphthylalkyl.

[0096] "Lower alkylene group" refers to a C1-C2 tether group such as a -CH2- tether group. 25"""" refers to a straight-chain alkyl tether group that forms a bond connecting molecular fragments through a terminal carbon atom. Examples include, but are not limited to, methylene (-CH2-), ethylene (-CH2CH2-), propylene (-CH2CH2CH2-), and butylene (-CH2CH2CH2CH2-). Lower alkylene groups can be substituted by replacing one or more hydrogens of the lower alkylene group with a substituent described in the definition of "substituted."

[0097] "Cycloalkyl" means a monocyclic or polycyclic hydrocarbon ring system that is fully saturated (no double or triple bonds). When composed of two or more rings, the rings may be fused together. A cycloalkyl group can contain 3 to 10 atoms, or 3 to 8 atoms, in the ring. A cycloalkyl group can be unsubstituted or substituted. Representative cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl.

[0098] "Cycloalkenyl" means a monocyclic or polycyclic hydrocarbon ring system containing one or more double bonds in at least one ring, provided that if there are two or more, the double bonds cannot form a completely delocalized pi-electron system through all rings (otherwise the group would be "aryl" as defined herein). When composed of two or more rings, the rings may be fused together. Cycloalkenyl groups can be unsubstituted or substituted.

[0099] "Cycloalkynyl" refers to a monocyclic or polycyclic hydrocarbon ring system containing one or more triple bonds in at least one ring. If there are more than one triple bond, the triple bonds cannot form a completely delocalized pi-electron system through all rings. When composed of two or more rings, the rings may be fused together. Cycloalkynyl groups may be unsubstituted or substituted.

[0100] "Alkoxy" or "alkyloxy" means a group of the formula -OR, where R is alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, or cycloalkynyl, as defined above. Examples of alkoxy include methoxy, ethoxy, n-propoxy, 1-methylethoxy (isopropoxy), n-butoxy, iso-butoxy, sec-butoxy, and tert-butoxy. Alkoxy can be substituted or unsubstituted.

[0101] "Acyl" refers to a group in which hydrogen, alkyl, alkenyl, alkynyl, aryl, or heteroaryl is bonded as a substituent via a carbonyl group such as -(C=O)-R. Examples include formyl, acetyl, propanoyl, benzoyl, and acryl. Acyl may be substituted or unsubstituted.

[0102] "Alkoxyalkyl" or "alkyloxyalkyl" means, as a substituent, an alkoxy group connected via a lower alkylene group. Examples include alkyl-O-alkyl- and alkoxy-alkyl-, where the terms alkyl and alkoxy are defined herein.

[0103] "Hydroxyalkyl" refers to an alkyl group in which one or more hydrogen atoms have been replaced with a hydroxy group. Exemplary hydroxyalkyl groups include, but are not limited to, 2-hydroxyethyl, 3-hydroxypropyl, 2-hydroxypropyl, and 2,2-dihydroxyethyl. Hydroxyalkyl can be substituted or unsubstituted.

[0104] "Haloalkyl" refers to an alkyl group in which one or more hydrogen atoms are replaced with halogen (e.g., monohaloalkyl, dihaloalkyl, trihaloalkyl). Examples include chloromethyl, fluoromethyl, difluoromethyl, trifluoromethyl, 1-chloro-2-fluoromethyl, 2-fluoroisobutyl, and the like. Haloalkyl can be substituted or unsubstituted.

[0105] "Amino" means "-NH2".

[0106] "Hydroxy" means "-OH".

[0107] "Cyano" means "-CN".

[0108] "Carbonyl" or "oxo" means "-C=O".

[0109] "Azido" means "-N3".

[0110] "Aminoalkyl" means, as a substituent, an amino group connected via a lower alkylene group. Examples include HN-alkyl-, with the term alkyl being defined herein.

[0111] "Alkylcarboxyalkyl" means an alkyl group bound as a substituent to a carboxy group which is bound as a substituent to an alkyl group. Examples include alkyl-(C=O)-O-alkyl- and alkyl-O-(C=O)-alkyl-, where the term alkyl is defined herein.

[0112] "Alkylaminoalkyl" means an alkyl group attached as a substituent to an amino group which is attached as a substituent to an alkyl group. Examples include alkyl-NH-alkyl-, where the term alkyl is as defined herein.

[0113] "Dialkylaminoalkyl" and "di(alkyl)aminoalkyl" refer to two alkyl groups bonded as substituents to an amino group, which is bonded as a substituent to an alkyl group, respectively. Examples include the term alkyl as defined herein. [ka] .

[0114] "Alkylaminoalkylamino" means an alkyl group attached, as a substituent, to an amino group. Examples include alkyl-NH-alkyl-NH-, where the term alkyl is defined herein.

[0115] "Alkylaminoalkylaminoalkylamino" means an alkyl group bound, as a substituent, to an amino group which is bound to an alkyl group. Examples include alkyl-NH-alkyl-NH-alkyl-, where the term alkyl is as defined herein.

[0116] "Arylaminoalkyl" means an aryl group bound as a substituent to an amino group which is bound as a substituent to an alkyl group. Examples include aryl-NH-alkyl-, where aryl and alkyl are defined herein.

[0117] "Aminoalkyloxy" means an amino group attached as a substituent to an alkyloxy group. Examples include HN-alkyl-O- and HN-alkoxy-, where the terms alkyl and alkoxy are defined herein.

[0118] "Aminoalkyloxyalkyl" means an amino group bound as a substituent to an alkyloxy group which is bound as a substituent to an alkyl group. Examples include HN-alkyl-O-alkyl- and HN-alkoxy-alkyl-, where the terms alkyl and alkoxy are defined herein.

[0119] "Aminoalkylcarboxy" means an amino group attached, as a substituent, to an alkyl group which is attached, as a substituent, to a carboxy group. Examples include HN-alkyl-(C=O)-O- and HN-alkyl-O-(C=O)-, with the term alkyl as defined herein.

[0120] "Aminoalkylaminocarbonyl" means an amino group bound, as a substituent, to an alkyl group which is bound to a carbonyl group. Examples include HN-alkyl-NH-(C=O)-, where the term alkyl is defined herein.

[0121] "Aminoalkylcarboxamido" means an amino group bound as a substituent to a carbonyl group bound as a substituent to an alkyl group bound as a substituent to the amino group. Examples include HN-alkyl-(C=O)-NH- and HN-alkyl-NH-(C=O)-, with the term alkyl as defined herein.

[0122] "Azidoalkyloxy" means an azido group attached as a substituent to an alkyloxy group. Examples include N3-alkyl-O- and N3-alkoxy-, where the terms alkyl and alkoxy are defined herein.

[0123] "Cyanoalkyloxy" means a cyano group attached as a substituent to an alkyloxy group. Examples include N-C-alkyl-O- and N-C-alkoxy-, where the terms alkyl and alkoxy are defined herein.

[0124] "Sulfenyl" refers to "-SR" where R is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, heteroalicyclyl, aralkyl, or (heteroalicyclyl)alkyl. The sulfenyl can be substituted or unsubstituted.

[0125] "Sulfinyl" means "-(S=O)-R", where R can be the same as defined for sulfenyl. The sulfinyl can be substituted or unsubstituted.

[0126] "Sulfonyl" means "-(S=O)-OR", where R can be the same as defined for sulfonyl. The sulfonyl can be substituted or unsubstituted.

[0127] "O-carboxy" means "R-(C=O)-O-," where R can be hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, heteroalicyclyl, aralkyl, or (heteroalicyclyl)alkyl, as defined herein. The O-carboxy can be substituted or unsubstituted.

[0128] "Ester" and "C-carboxy" refer to "-(C=O)-OR", where R can be the same as defined for O-carboxy. Ester and C-carboxy can be substituted or unsubstituted.

[0129] "Thiocarbonyl" means "-(C=S)-R", where R can be the same as defined for O-carboxy. The thiocarbonyl can be substituted or unsubstituted.

[0130] "Trihalomethanesulfonyl" refers to "X3CSO2-" where X is a halogen.

[0131] "S-sulfonamide" refers to "-SON(RARB)," where R and R can independently be hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, heteroalicyclyl, aralkyl, or (heteroalicyclyl)alkyl. S-sulfonamides can be substituted or unsubstituted.

[0132] "N-sulfonamide" means "RSON(RA)-", where R and R can independently be hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, heteroalicyclyl, aralkyl, or (heteroalicyclyl)alkyl. N-sulfonamide can be substituted or unsubstituted.

[0133] "O-carbamyl" and "urethanyl" refer to "-O-(C=O)-N(RARB)," where R and R can independently be hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, heteroalicyclyl, aralkyl, or (heteroalicyclyl)alkyl. O-carbamyl or urethanyl can be substituted or unsubstituted.

[0134] "N-carbamyl" means "RO-(C=O)-N(RA)-," where R and R can independently be hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, heteroalicyclyl, aralkyl, or (heteroalicyclyl)alkyl. The N-carbamyl can be substituted or unsubstituted.

[0135] "O-thiocarbamyl" refers to "-O-(C=S)-N(RARB)," where R and R can independently be hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, heteroalicyclyl, aralkyl, or (heteroalicyclyl)alkyl. The O-thiocarbamyl can be substituted or unsubstituted.

[0136] "N-thiocarbamyl" means "RO-(C=S)-N(RA)-," where R and R can independently be hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, heteroalicyclyl, aralkyl, or (heteroalicyclyl)alkyl. N-thiocarbamyl can be substituted or unsubstituted.

[0137] "C-amido" means "-(C=O)-N(RARB)" where R and R are independently hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, heteroalicyclyl, aralkyl, or (heteroalicyclyl)alkyl. C-amido can be substituted or unsubstituted.

[0138] "N-amido" means "R-(C=O)-N(RA)-", where R and R are independently hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, heteroalicyclyl, aralkyl, or (heteroalicyclyl)alkyl. N-amido can be substituted or unsubstituted.

[0139] "Guanidinoalkyloxy" refers to a guanidinyl group attached as a substituent to an alkyloxy group. Examples include: [ka] , including the terms alkyl and alkoxy as defined herein.

[0140] "Guanidinoalkylcarboxy" refers to a guanidinyl group attached as a substituent to an alkyl group which is attached to a carboxy group. Examples include: [ka] , and the term alkyl as defined herein.

[0141] "Quaternary ammonium alkyl carboxy" refers to a compound in which a quaternized amino group is bonded as a substituent to an alkyl group which is bonded to a carboxy group. Examples include: [ka] , and the term alkyl as defined herein.

[0142] "Halogen atom" and "halogen" mean any of the radiation-stable atoms listed in column 7 of the periodic table of the elements, including, for example, fluorine, chlorine, bromine, and iodine.

[0143] Where the number of substituents is not specified (e.g., haloalkyl), one or more substituents may be present. For example, "haloalkyl" may include one or more halogens which may be the same or different.

[0144] "Amino acid" refers to any amino acid (both standard and non-standard), including, but not limited to, α-amino acids, β-amino acids, γ-amino acids, and δ-amino acids. Examples of suitable amino acids include, but are not limited to, alanine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, proline, serine, tyrosine, arginine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine. Additional examples of suitable amino acids include, but are not limited to, ornithine, hypusine, 2-aminoisobutyric acid, dehydroalanine, γ-aminoisobutyric acid, citrulline, β-alanine, α-ethylglycine, α-propylglycine, and norleucine.

[0145] A "linking group" is a divalent moiety used to link one steroid to another. In embodiments, a linking group is used to link a first CSA to a second CSA (which may be the same or different). Examples of linking groups include (C1-C 10 )Alkyloxy-(C1-C 10 ) alkyl.

[0146] "PG" or "protecting group" or "protecting group" means an atom or group of atoms added to a molecule to prevent existing groups in the molecule from undergoing undesired chemical reactions. Examples of protecting group moieties are described in T.W. Greene and P.G.M. Buts, Protective Groups in Organic Synthesis, 3. Ed. John Wiley & Sons, 1999, and J.F.W.M. McOmie, Protective Groups in Organic Chemistry Plenum Press, 1973, both of which are incorporated herein by reference for the limited purpose of disclosing suitable protecting groups. Protecting groups can be selected to be stable to particular reaction conditions and easily removed at a convenient stage using methodologies known in the art.A non-limiting list of protecting groups includes benzyl, substituted benzyl, alkylcarbonyl and alkoxycarbonyl (e.g., t-butoxycarbonyl (BOC), acetyl, or isobutyryl), arylalkylcarbonyl and arylalkoxycarbonyl (e.g., benzyloxycarbonyl); substituted methyl ethers (e.g., methoxymethyl ether); substituted ethyl ethers; substituted benzyl ethers; tetrahydropyranyl ethers; silyls (e.g., trimethylsilyl, triethylsilyl, triisopropylsilyl, t-butyldimethylsilyl, triisopropylsilyloxymethyl, [2-(trimethylsilyl)ethoxy]methyl, or t-butyldiphenylsilyl, etc.); esters (e.g., benzoates, etc.); carbonyl ethers (e.g., benzoates, etc.); Examples of protecting groups include acid salts (e.g., methoxymethyl carbonate, etc.); sulfonates (e.g., tosylate or mesylate, etc.); acyclic ketals (e.g., dimethyl acetal, etc.); cyclic ketals (e.g., 1,3-dioxane, 1,3-dioxolane, and those described herein); acyclic acetals; cyclic acetals (e.g., those described herein); acyclic hemiacetals; cyclic hemiacetals; cyclic dithioketals (e.g., 1,3-dithiane or 1,3-dithiolane); orthoesters (e.g., those described herein); and triarylmethyl groups (e.g., trityl; monomethoxytrityl (MMTr); 4,4'-dimethoxytrityl (DMTr); 4,4',4''-trimethoxytrityl (TMTr); and those described herein). Amino protecting groups are known to those skilled in the art. Generally, the type of protecting group is not critical, is stable to the conditions of subsequent reactions at other positions in the compound, and can be removed at the appropriate time without adversely affecting the remainder of the molecule. Furthermore, protecting groups can be replaced with other protecting groups after substantial synthetic reactions are complete. Clearly, a compound is within the scope of this disclosure if it differs from a compound disclosed herein only in that one or more protecting groups of the disclosed compound have been replaced with a different protecting group.

[0147] The present invention may be embodied in other specific forms without departing from its spirit or essential characteristics. The described embodiments are to be considered in all respects only as illustrative and not restrictive. The scope of the invention is, therefore, indicated by the appended claims rather than by the foregoing description. All changes that come within the meaning and range of equivalency of the claims are to be embraced within their scope.

Claims

1. 1. A method for preventing tissue regeneration and / or tissue atrophy, comprising: providing a therapeutic composition comprising one or more CSA compounds in a carrier; administering the therapeutic composition to a target tissue of a subject; wherein the therapeutic composition stimulates tissue regeneration and / or prevents tissue atrophy in the target tissue to which it is applied; A method comprising:

2. 10. The method of claim 1, wherein the therapeutic composition comprises one or more CSA compounds having a hydrolyzable bond.

3. The method of claim 2, wherein the CSA compound contained in the therapeutic composition comprises a majority by weight of a CSA compound having a hydrolyzable bond.

4. 4. The method of claim 3, wherein substantially all of the CSA compounds contained in the therapeutic composition are CSA compounds having hydrolyzable bonds.

5. The method according to any one of claims 2 to 4, wherein the hydrolyzable bond is an ester bond.

6. 6. The method of any one of claims 1 to 5, wherein the one or more CSA compounds comprise one or more of CSA-44, CSA-142, CSA-144, CSA-145, CSA-146, and CSA-148.

7. 7. The method of any one of claims 1 to 6, wherein the treatment composition is applied to a dermal area and stimulates hair growth and / or prevents hair loss in the dermal area to which the treatment composition is applied.

8. 8. The method of claim 7, wherein the dermal region is affected by a microbial infection associated with hair loss in the dermal region.

9. 9. The method of claim 8, wherein the microbial infection is a dermatophyte infection.

10. 10. The method of claim 8 or 9, wherein the therapeutic composition kills or inactivates the microorganism causing the underlying infection.

11. 11. The method of claim 10, wherein at least a portion of the hair stimulating and / or hair loss preventing activity of the treatment composition is independent of the antimicrobial activity of the treatment composition.

12. 12. The method of any one of claims 1 to 11, wherein the subject is a human, a farm animal, a pet, a laboratory animal, or a zoo animal.

13. 13. The method of any one of claims 7 to 12, wherein the dermal area is affected by or at risk of being affected by hair loss as a result of one or more of an infection, a genetic disorder, a hormonal imbalance, or an injury.

14. 14. The method of any one of claims 7 to 13, wherein the subject is a human and the area of ​​skin is the scalp.

15. 15. The method of any one of claims 1 to 14, wherein the carrier is selected from the group consisting of water, alcohol, dimethyl sulfoxide, an organic solvent, an emulsion, and combinations thereof.

16. 16. The method of any one of claims 1 to 15, wherein the therapeutic composition is provided in the form of a liniment, lotion, ointment, cream, powder, wash, or spray.

17. 16. The method of any one of claims 1 to 15, wherein the treatment composition is incorporated into a shampoo, conditioner, or hair care product.

18. 18. The method of any one of claims 1-17, wherein the treatment composition restores hair growth at about 1.2 to 5 times, or about 1.5 to 3 times, the rate of hair growth in the absence of CSA-based treatment.

19. 7. The method of any one of claims 1-6, wherein the target tissue is tissue damaged as a result of cancer treatment, stroke, osteoarthritis, autoimmune disease, spinal cord injury, brain injury, cardiac injury or disorder, or type 1 diabetes.

20. 20. The method of any one of claims 1 to 19, wherein the therapeutic composition is applied directly to a target tissue to stimulate local stem cells.

21. 21. The method of claim 20, wherein the local stem cells comprise one or more of follicular stem cells, hematopoietic stem cells, neural stem cells, epithelial stem cells of the intestine or skin, or tissue stem cells found in bone marrow, peripheral blood, brain, spinal cord, dental pulp, blood vessels, skeletal muscle, cornea, retina, liver, and pancreas.

22. 22. The method of any one of claims 1 to 21, wherein the treatment is performed without separately collecting, mixing, or culturing stem cells.

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