COMPOSITIONS AND METHODS FOR MODULATING HAIR GROWTH

The present disclosure relates to pharmaceutical compositions containing electron transport chain (ETC) inhibitors, which are capable of promoting hair growth. The disclosure further relates to methods of promoting hair growth or treating conditions or disorders affecting hair growth, such as baldness or alopecia.

BACKGROUND

Hair follicle stem cells (HFSCs) undergo successive rounds of quiescence (telogen) punctuated by brief periods of proliferation correlating with the start of the hair cycle (telogen-anagen transition). Proliferation or activation of HFSCs is well known to be a prerequisite for advancement of the hair cycle. Despite advances in treatment options, baldness and alopecia continue to be conditions that cannot be successfully treated in all individuals. Some of the existing treatments are inconvenient for users, others require surgical intervention or other invasive procedures. Additional therapies are needed.

SUMMARY OF THE INVENTION

In certain aspects, the present disclosure provides pharmaceutical compositions comprising inhibitors of the Electron Transport Chain (ETC). In certain embodiments, the pharmaceutical compositions are formulated for topical administration.

In certain aspects, the present disclosure provides methods of promoting hair growth, comprising administering to a patient a therapeutically effective amount of a composition as described herein.

DETAILED DESCRIPTION OF THE INVENTION

While many signaling pathways have been implicated in control of activation of adult hair follicle stem cells (HFSCs) and the hair cycle, less is known about cell intrinsic mechanisms of stem cell control. Lactate production has been identified as a key cell intrinsic regulator of hair follicle stem cell activity, suggesting that cellular metabolism is important in stem cell activation. Transgenic methods have been used to suggest that transgenic blockade of the Electron Transport Chain (ETC) leads to degeneration of the hair follicle. However, the present disclosure provides composition and methods by which pharmacological abrogation of ETC activity, as opposed to complete ablation of ETC, can promote hair cycle activation without significant cell toxicity. Furthermore, the metabolic data provided herein suggest that ETC inhibition leads to increased pyruvate accessibility for the Ldh enzyme and therefore increased lactate production, which can promote hair cycle activation. Finally, this type of ETC inhibition can even be used to accelerate the hair cycle in aged mice. These results point toward an unexpected and safe method to promote hair follicle stem cell activation.

Over the last three decades, a number of signaling pathways have been identified that act on HFSCs to promote both quiescence as well as their activation. With respect to intrinsic mechanisms of HFSC regulation, less is known about the cellular metabolism of individual cell types in the epidermis. In general, it has been presumed that somatic cells use mostly the electron transport chain (ETC) to produce energy from pyruvate that was generated by the uptake and processing of glucose, while early embryonic and cancer cells are thought to also rely on production of lactate from pyruvate. HFSCs balance the production of energy through the ETC with the production of lactate as well. Previous efforts to define metabolic activities in the epidermis focused on measurements of enzyme activities on entire follicles. In addition, several studies used transgenic models targeting the entire epidermis (including the follicle) for deletion of ETC components. Those studies suggested that genetic blockade of the ETC leads to degeneration of the follicle. However, it is not clear whether inhibition of ETC complexes—as opposed to genetic ablation of ETC complexes—would affect cell metabolism or fate decisions.

The present disclosure shows that inhibiting ETC activity causes proliferation of HFSCs and promotes hair growth. As used herein, the term “ETC inhibitor” includes any agent that is capable of inhibiting ETC complexes I, II, III, or IV, preferably ETC complexes I or III. Inhibitors of each of these complexes are known in the art. Inhibitors of ETC complex I include metformin, phenformin, buformin, rotenone, epiberberine, piericidin A, amytal, capsaicin, haloperidol, risperidone, bupivacaine, lidocaine, halothane, dantrolene, phenyloin, clofibrate, and fenofibrat. Inhibitors of ETC complex II include sodium malonate, thenoyltrifluoroacetone, cyclophosphamide, and ketoconazole. Inhibitors of ETC complex III include antimycin A, acetaminophen, isoflurane, and sevoflurane. Inhibitors of ETC complex IV include cephaloridine, cefazolin, and cefalotin. Certain ETC inhibitors are generally described in U.S. Pat. No. 8,993,587, which is hereby incorporated by reference as if fully set forth herein.

In certain aspects, the present disclosure provides pharmaceutical compositions formulated for topical administration comprising inhibitors of the Electron Transport Chain (ETC). As described herein, ETC inhibitors cause proliferation of HFSCs and can thereby promote hair growth.

In certain embodiments, the electron transport chain inhibitor is an inhibitor of electron transport chain complex I or III. In certain embodiments, the electron transport chain inhibitor is metformin, phenformin, buformin, rotenone, epiberberine, piericidin A, amytal, capsaicin, haloperidol, risperidone, bupivacaine, lidocaine, halothane, dantrolene, phenyloin, clofibrate, fenofibrat, antimycin A, acetaminophen, isoflurane, or sevoflurane. In certain embodiments, the electron transport chain inhibitor is rotenone, phenformin, or antimycin A.

In certain aspects, the present disclosure provides methods of promoting hair growth, comprising administering to a patient a therapeutically effective amount of a composition comprising an ETC inhibitor as described herein. In certain embodiments, the condition or disorder is baldness or alopecia.

Pharmaceutical Compositions

The compositions and methods of the present invention may be utilized to treat an individual in need thereof. In certain embodiments, the individual is a mammal such as a human, or a non-human mammal. When administered to an animal, such as a human, the composition or the compound is preferably administered as a pharmaceutical composition comprising, for example, a compound as disclosed herein and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are well known in the art and include, for example, aqueous solutions such as water or physiologically buffered saline or other solvents or vehicles such as glycols, glycerol, oils such as olive oil, or injectable organic esters. In preferred embodiments, when such pharmaceutical compositions are for human administration, particularly for invasive routes of administration (i.e., routes, such as injection or implantation, that circumvent transport or diffusion through an epithelial barrier), the aqueous solution is pyrogen-free, or substantially pyrogen-free. The excipients can be chosen, for example, to effect delayed release of an agent or to selectively target one or more cells, tissues or organs. The pharmaceutical composition can be in dosage unit form such as tablet, capsule (including sprinkle capsule and gelatin capsule), granule, lyophile for reconstitution, powder, solution, syrup, suppository, injection or the like. The composition can also be present in a transdermal delivery system, e.g., a skin patch. The composition can also be present in a solution suitable for topical administration, such as a lotion, cream, or ointment.

A pharmaceutical composition (preparation) can be administered to a subject by any of a number of routes of administration including, for example, orally (for example, drenches as in aqueous or non-aqueous solutions or suspensions, tablets, capsules (including sprinkle capsules and gelatin capsules), boluses, powders, granules, pastes for application to the tongue); absorption through the oral mucosa (e.g., sublingually); subcutaneously; transdermally (for example as a patch applied to the skin); and topically (for example, as a cream, ointment or spray applied to the skin). The compound may also be formulated for inhalation. In certain embodiments, a compound may be simply dissolved or suspended in sterile water. Details of appropriate routes of administration and compositions suitable for same can be found in, for example, U.S. Pat. Nos. 6,110,973, 5,763,493, 5,731,000, 5,541,231, 5,427,798, 5,358,970 and 4,172,896, as well as in patents cited therein.

Methods of preparing these formulations or compositions include the step of bringing into association an active compound, such as a compound as disclosed herein, with the carrier and, optionally, one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association a compound as disclosed herein with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.

The phrases “parenteral administration” and “administered parenterally” as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion. Pharmaceutical compositions suitable for parenteral administration comprise one or more active compounds in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.

For use in the methods of this invention, active compounds can be given per se or as a pharmaceutical composition containing, for example, 0.1 to 99.5% (more preferably, 0.5 to 90%) of active ingredient in combination with a pharmaceutically acceptable carrier.

A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the therapeutically effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the pharmaceutical composition or compound at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. By “therapeutically effective amount” is meant the concentration of a compound that is sufficient to elicit the desired therapeutic effect. It is generally understood that the effective amount of the compound will vary according to the weight, sex, age, and medical history of the subject. Other factors which influence the effective amount may include, but are not limited to, the severity of the patient's condition, the disorder being treated, the stability of the compound, and, if desired, another type of therapeutic agent being administered with the compound as disclosed herein. A larger total dose can be delivered by multiple administrations of the agent. Methods to determine efficacy and dosage are known to those skilled in the art (Isselbacher et al. (1996) Harrison's Principles of Internal Medicine 13 ed., 1814-1882, herein incorporated by reference).

The patient receiving this treatment is any animal in need, including primates, in particular humans; and other mammals such as equines, cattle, swine, sheep, cats, and dogs; poultry; and pets in general.

In certain embodiments, compounds of the invention may be used alone or conjointly administered with another type of therapeutic agent.

The pharmaceutically acceptable acid addition salts can also exist as various solvates, such as with water, methanol, ethanol, dimethylformamide, and the like. Mixtures of such solvates can also be prepared. The source of such solvate can be from the solvent of crystallization, inherent in the solvent of preparation or crystallization, or adventitious to such solvent.

Definitions

Unless otherwise defined herein, scientific and technical terms used in this application shall have the meanings that are commonly understood by those of ordinary skill in the art. Generally, nomenclature used in connection with, and techniques of, chemistry, cell and tissue culture, molecular biology, cell and cancer biology, neurobiology, neurochemistry, virology, immunology, microbiology, pharmacology, genetics and protein and nucleic acid chemistry, described herein, are those well known and commonly used in the art.

The methods and techniques of the present disclosure are generally performed, unless otherwise indicated, according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout this specification. See, e.g. “Principles of Neural Science”, McGraw-Hill Medical, New York, N.Y. (2000); Motulsky, “Intuitive Biostatistics”, Oxford University Press, Inc. (1995); Lodish et al., “Molecular Cell Biology, 4th ed.”, W. H. Freeman & Co., New York (2000); Griffiths et al., “Introduction to Genetic Analysis, 7th ed.”, W. H. Freeman & Co., N.Y. (1999); and Gilbert et al., “Developmental Biology, 6th ed.”, Sinauer Associates, Inc., Sunderland, Mass. (2000).

Chemistry terms used herein, unless otherwise defined herein, are used according to conventional usage in the art, as exemplified by “The McGraw-Hill Dictionary of Chemical Terms”, Parker S., Ed., McGraw-Hill, San Francisco, Calif. (1985).

All of the above, and any other publications, patents and published patent applications referred to in this application are specifically incorporated by reference herein. In case of conflict, the present specification, including its specific definitions, will control.

The term “agent” is used herein to denote a chemical compound (such as an organic or inorganic compound, a mixture of chemical compounds), a biological macromolecule (such as a nucleic acid, an antibody, including parts thereof as well as humanized, chimeric and human antibodies and monoclonal antibodies, a protein or portion thereof, e.g., a peptide, a lipid, a carbohydrate), or an extract made from biological materials such as bacteria, plants, fungi, or animal (particularly mammalian) cells or tissues. Agents include, for example, agents whose structure is known, and those whose structure is not known.

A “patient,” “subject,” or “individual” are used interchangeably and refer to either a human or a non-human animal. These terms include mammals, such as humans, primates, livestock animals (including bovines, porcines, etc.), companion animals (e.g., canines, felines, etc.) and rodents (e.g., mice and rats).

“Treating” a condition or patient refers to taking steps to obtain beneficial or desired results, including clinical results. As used herein, and as well understood in the art, “treatment” is an approach for obtaining beneficial or desired results, including clinical results. Beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of extent of disease, stabilized (i.e. not worsening) state of disease, preventing spread of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. “Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment.

The term “preventing” is art-recognized, and when used in relation to a condition, such as a local recurrence (e.g., pain), a disease such as cancer, a syndrome complex such as heart failure or any other medical condition, is well understood in the art, and includes administration of a composition which reduces the frequency of, or delays the onset of, symptoms of a medical condition in a subject relative to a subject which does not receive the composition. Thus, prevention of cancer includes, for example, reducing the number of detectable cancerous growths in a population of patients receiving a prophylactic treatment relative to an untreated control population, and/or delaying the appearance of detectable cancerous growths in a treated population versus an untreated control population, e.g., by a statistically and/or clinically significant amount.

“Administering” or “administration of” a substance, a compound or an agent to a subject can be carried out using one of a variety of methods known to those skilled in the art. For example, a compound or an agent can be administered, intravenously, arterially, intradermally, intramuscularly, intraperitoneally, subcutaneously, ocularly, sublingually, orally (by ingestion), intranasally (by inhalation), intraspinally, intracerebrally, and transdermally (by absorption, e.g., through a skin duct). A compound or agent can also appropriately be introduced by rechargeable or biodegradable polymeric devices or other devices, e.g., patches and pumps, or formulations, which provide for the extended, slow or controlled release of the compound or agent. Administering can also be performed, for example, once, a plurality of times, and/or over one or more extended periods.

Appropriate methods of administering a substance, a compound or an agent to a subject will also depend, for example, on the age and/or the physical condition of the subject and the chemical and biological properties of the compound or agent (e.g., solubility, digestibility, bioavailability, stability and toxicity). In some embodiments, a compound or an agent is administered orally, e.g., to a subject by ingestion. In some embodiments, the orally administered compound or agent is in an extended release or slow release formulation, or administered using a device for such slow or extended release.

As used herein, the phrase “conjoint administration” refers to any form of administration of two or more different therapeutic agents such that the second agent is administered while the previously administered therapeutic agent is still effective in the body (e.g., the two agents are simultaneously effective in the patient, which may include synergistic effects of the two agents). For example, the different therapeutic compounds can be administered either in the same formulation or in separate formulations, either concomitantly or sequentially. Thus, an individual who receives such treatment can benefit from a combined effect of different therapeutic agents.

A “therapeutically effective amount” or a “therapeutically effective dose” of a drug or agent is an amount of a drug or an agent that, when administered to a subject will have the intended therapeutic effect. The full therapeutic effect does not necessarily occur by administration of one dose, and may occur only after administration of a series of doses. Thus, a therapeutically effective amount may be administered in one or more administrations. The precise effective amount needed for a subject will depend upon, for example, the subject's size, health and age, and the nature and extent of the condition being treated, such as cancer or MDS. The skilled worker can readily determine the effective amount for a given situation by routine experimentation.

Examples

Example 1: Effect of ETC Activity on HFSC Activation

To determine whether manipulation of ETC activity could affect HFSC activation, various inhibitors of ETC components were topically applied to mice during a resting phase of the hair cycle. The topical formulation was prepared by suspending the active ingredient in PLO Ultramax Gel (lecithin organogel). At postnatal day 50, the hair follicle is in telogen, a resting phase where the stem cells of the follicle are quiescent until the start of the next hair cycle at day 70-80. Rotenone, Phenformin, and Antimycin A are established inhibitors of Complex I, and Complex III, respectively. Animals were shaved at postnatal day 47 and treated with the indicated compounds or vehicle on the shaved area every 48 hours for the indicated duration. After 3-4 treatments (8-12 days), animals treated with ETC inhibitors began to show signs of hair cycle activation macroscopically, judged by pigmentation of the skin in black mice whereas vehicle treated mice did not show significant pigmentation for at least 20 days (FIGS. 1A and 3A). The epidermis of murine skin becomes pigmented upon induction of the hair cycle, which is indicative of the generation of melanocytes injecting pigment (melanin) into the keratinocytes that go on to make the hair shaft, as well as those in the interfollicular epidermis. Therefore, the induction of pigmentation observed after 8-12 days in ETC inhibitor treated mice was most likely indicative of hair cycle activation induced by this treatment.

Example 2: Pathology of ETC-Inhibited Tissues

To demonstrate that the pigmentation induced by ETC inhibition was in fact due to changes in hair follicle stem cell activation, tissue was harvested and subjected to pathology. Histological analysis showed that follicles in backskin treated with ETC inhibitors promoted a normal telogen-to-anagen transition (FIGS. 1B and 3B). These findings were also in stark contrast to previous studies showing that transgenic abrogation of the ETC led to hair follicle degeneration.

Example 3: Skin Thickness Measurements

To determine whether the hair cycle induction driven by ETC inhibition was typical, the thickness of each layer of skin was measured at different stages of treatment. As shown inFIG. 1C, all of the ETC inhibitors increased the thickness of the epidermis, dermis, and particularly the hypodermis, suggesting a strong expansion of the adipocytes. Analysis of ETC inhibited skin showed a strong increase in Ki67 in HFSCs a week after treatment, evidence of HFSC activation in response to ETC inhibition (FIGS. 1D and 3D). To determine whether application of the ETC inhibitors promoted inflammation, which could cloud interpretation of hair cycle data, various markers of chemokine response and the presence of inflammatory immune cells were assessed after treatment. There was no evidence of significant inflammation by these measures in response to ETC inhibition (FIG. 3D).

Example 4: Metabolic Measurements

To determine the effect on cellular metabolism of ETC inhibition by Rotenone, Phenformin and Antimycin A, two measures of metabolic pathways were performed. First, LDH activity was quantified on cells isolated from the epidermis treated with ETC inhibitors for 48 hours (FIG. 2A). Next, metabolomics was employed on sorted HFSCs with and without treatment for either 48 hours or 10 days. These analyses indicated an increase in lactate levels as well as several other glycolytic intermediates in response to ETC inhibition by Rotenone, Phenformin and Antimycin A (FIG. 2B).

Example 5: Effect of ETC Inhibition on Aged Mice

As mice age, the hair cycle is known to become protracted such that upon shaving, only portions of the backskin show regrowth of hair within a 1-2 months. Various batches of aged mice (at least 17 months) were treated for 30 days with ETC inhibitors to determine whether this metabolic manipulation could stimulate the hair cycle even in dormant follicles. Topical application of Phenformin, Rotenone or Antimycin A all led to more complete hair regrowth across the entire backskin on a similar time course to that of younger mice (FIG. 4A). As in younger animals, treatment with these ETC inhibitors led to an increase in lactate pool levels as measured by metabolomics (FIG. 4B).

INCORPORATION BY REFERENCE

EQUIVALENTS