Methods for hair regeneration

Abstract

The present invention relates to pharmaceutical compositions and methods for stimulating hair growth and / or restoring hair pigmentation in a subject and the compositions comprising extracellular vesicles related to such methods.

Description

Atty Docket No.: KIBS-001 / 01WO 357284-2006METHODS FOR HAIR REGENERATIONCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 761,320, filed February 21, 2025, the disclosure of which is herein incorporated by reference in its entirety.BACKGROUND OF THE INVENTION

[0002] Hair loss is a medical abnormality or disease that affects over 40 million men in America and a substantial amount of women as well (Geraci R.; Men's Health, June 1999). Hair loss is commonly divided into two categories, cicatricial (scarring) and non-ciacatricial alopecia. The cicatricial alopecia results from hair follicle damage complicated by various pathological changes in the surrounding skin, in which burns are a common cause. Non-cicatricial alopecia is caused by either functional or structural disorders of the hair follicle itself. This latter condition may be further divided into primary and secondary causes. Secondary follicular disorders are usually the results of chemotherapy or radiation treatments for cancer, nutritional, hormonal disorders or even stress. Alopecia areata and male pattern alopecia (androgenetica alopecia) are common primary follicular disorders, in which androgenetica alopecia (ADA) is the most common, causing refractory and mostly irreversible baldness. Their fully pathogenetic mechanisms are unknown.

[0003] Hair growth is a dynamic process involving phases or steps; each and every hair grows from an individual hair follicle, the size and length of the follicle determines the thickness and relative length of the hair. Under normal healthy conditions the normal pattern of body hair growth is generally well maintained with a balance of terminal hairs (coarse and long) and vellus hairs (thin and short). In the presence of hormonal abnormalities or changes, inflammation, toxic exposures, and stress, abnormal conditions within the follicle may occur, inducing either gradual thinning or rapid loss of the hair.

[0004] It has previously been disclosed that the rate and character of hair growth can be altered by applying to the skin inhibitors of certain enzymes. These inhibitors include inhibitors of 5-alpha reductase, ornithine decarboxylase, S-adenosylmethionine decarboxylase, gamma-glutamyl transpeptidase, and transglutaminase. See, for example, Hreuer et al., U. S. Pat. No. 4,885,289; Shander, U. S. Pat. No. 4,720,489; Ahluwalia, U. S. Pat. No. 5,095,007; Ahluwalia etal., U. S. Pat.Atty Docket No.: KIBS-001 / 01WO 357284-2006No. 5,096,911; Shander etal., U. S. Pat. No. 5,132,293; and Shander etal., U. S. Pat. No. 5,143,925. However, these inhibitors, as with many other potential treatments, are not particularly effective and may have adverse effects.

[0005] Graying of hair is also an issue in both men and women. Conventionally, hair dyeing has been mainly performed as a method of changing hair to black hair against graying due to aging. In recent years, however, it has been found that gray hair occurs when the function of hair pigment cells in the hair follicles of the scalp decreases due to aging or stress. However, there are still not sufficient cures or methods to prevent and improve gray hair. The emergence of materials having the effect of preventing and improving gray hair has been desired. New therapies are thus currently needed for both baldness and gray hair, or lack of hair pigmentation,BRIEF DESCRIPTION OF THE DRAWINGS

[0006] Fig. 1 shows a patient’s hair just before treatment with an extracellular vesicle composition and LIFU

[0007] Fig. 2 shows a patient’s hair 4 months after a single treatment with an extracellular vesicle composition and LIFU.SUMMARY OF THE INVENTION

[0008] The present invention relates to methods of stimulating hair growth and / or restoring hair pigmentation in a subject and administering pharmaceutical compositions. In a specific embodiment, the methods comprise stimulating hair growth and / or restoring hair pigmentation in a subject, comprising: a) injecting an extracellular vesicle into the subject; and b) administering an acoustic wave to the brain of the subject.

[0009] In a specific embodiment, the subject restores original pigmentation to their hair follicles. In another embodiment, the subject’s hair pigmentation is no longer white or gray. In another embodiment, the methods relate to the prevention of hair loss and / or the prevention of gray or white hair. In another specific embodiment, the restoration of pigmentation is on the subject’s scalp or head.

[0010] In another specific embodiment, the methods administration is when the subject is in deep sleep or REM sleep. In a specific embodiment, the subject is human.Atty Docket No.: KIBS-001 / 01WO 357284-2006

[0011] In another specific embodiment, the acoustic wave has a frequency of between about 500 khz to about 1,000 khz.

[0012] In another specific embodiment, the extracellular vesicle is an exosome, ranging from about 10 nm diameter to about 500 nm diameter. In another embodiment, the exosome has a diameter of about 40 nm to about 220 nm. In another specific embodiment, the extracellular vesicle or exosome is harvested from mesenchymal stem cells. In another embodiment, the mesenchymal stem cells are placental mesenchymal stem cells. In another specific embodiment, the extracellular vesicle is administered to the subject topically, trans dermally, parenterally, intradermally, intramuscularly, intraperitoneally, intravenously, subcutaneously, or by intranasal instillation, by intracavitary or intravesical instillation, intraocularly, intraarterially, intralesionally, or by application to mucous membranes. In another specific embodiment, the administration is intravenously to the subject.

[0013] In another specific embodiment, the subject’s brain that is subjected to the acoustic wave is selected from the group consisting of frontal lobe, parietal lobe, occipital lobe, temporal lobe, hippocampus, hypothalamus, brain stem, cerebellum amygdala, corticospinal tract, thalamus, substantia nigra, a basal ganglia and nasal submucosal injection around the inferior turbinates. In another embodiment, the extracellular vesicle or exosome enters into the brain by crossing the blood-brain barrier. In another specific embodiment, the acoustic wave is specifically focused on the hypothalamus of the subject. In another embodiment, prior to administration of acoustic wave to the brain, the subject is subjected to magnetic resonance imaging to determine the location of the hypothalamus or other desired portion of the brain of the subject.

[0014] In another embodiment, the acoustic wave is produced by a sonic transducer or an ultrasonic transducer, or an ultrasound device. In another specific embodiment, the acoustic wave administered to the subject’s brain is administered between 30 minutes to 4 hrs. In another embodiment, the acoustic wave administered to the subject’s brain is administered between 1 hr to 2 hrs.

[0015] In another embodiment, the administration of the acoustic wave is by Low intensity focused ultrasound (LIFU) or High-Intensity Focused Ultrasound (HIFU).

[0016] In another specific embodiment, the steps further comprise the step of administering a microbubble to the patient’s blood stream. In another embodiment, the methods include the comprising the additional step of using the sonic transducer to sonicate the microbubble. TheAtty Docket No.: KIBS-001 / 01WO 357284-2006present invention also includes pharmaceutical compositions comprising isolated extracellular vesicle exosomes, for example exosomes that comprise CD63+ / CD81+ surface markers and at least one pharmaceutically acceptable excipient. In another embodiment, the pharmaceutical composition additionally comprises isolated extracellular vesicles that are CD9+ / CD73+ microvesicles. In another specific embodiment, the isolated exosomes and microvesicles are in a heterogeneous mixture.DETAILED DESCRIPTION

[0017] Throughout this disclosure, various patents, patent applications and publications are referenced. The disclosures of these patents, patent applications and publications in their entireties are incorporated into this disclosure by reference for all purposes in order to more fully describe the state of the art as known to those skilled therein as of the date of this disclosure. This disclosure will govern in the instance that there is any inconsistency between the patents, patent applications and publications cited and this disclosure.

[0018] All publications, patents and patent applications, including any drawings and appendices therein are incorporated by reference in their entirety for ail purposes to the same extent as if each individual publication, patent or patent application, drawing, or appendix was specifically and individually indicated to be incorporated by reference in While the following terms are believed to be well understood by one of ordinary skill in the art, the following definitions are set forth to facilitate explanation of the presently disclosed subject matter.Definitions

[0019] Throughout the present specification, the terms “about” and / or “approximately” may be used in conjunction with numerical values and / or ranges. The term “about” is understood to mean those values near to a recited value. Furthermore, the phrases “less than about [a value]” or “greater than about [a value]” should be understood in view of the definition of the term “about” provided herein. The terms “about” and “approximately” may be used interchangeably.

[0020] Throughout the present specification, numerical ranges are provided for certain quantities. It is to be understood that these ranges comprise all subranges therein. Thus, the range “from 50Atty Docket No.: KIBS-001 / 01WO 357284-2006to 80” includes all possible ranges therein (e.g., 51-79, 52-78, 53-77, 54-76, 55-75, 60-70, etc.). Furthermore, all values within a given range may be an endpoint for the range encompassed thereby (e.g., the range 50-80 includes the ranges with endpoints such as 55-80, 50-75, etc.).

[0021] The term “a” or “an” refers to one or more of that entity; for example, “a cancer therapeutic” refers to one or more cancer therapeutics or at least one cancer therapeutic. As such, the terms “a” (or “an”), “one or more” and “at least one” are used interchangeably herein. In addition, reference to “an inhibitor” by the indefinite article “a” or “an” does not exclude the possibility that more than one of the inhibitors is present, unless the context clearly requires that there is one and only one of the inhibitors.

[0022] As used herein, the verb “comprise” as is used in this description and in the claims and its conjugations are used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. The present disclosure may suitably “comprise”, “consist of”, or “consist essentially of’, the steps, elements, and / or reagents described in the claims.

[0023] It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as "solely", "only" and the like in connection with the recitation of claim elements, or the use of a "negative" limitation.

[0024] The term "treating" means one or more of relieving, alleviating, delaying, reducing, improving, or managing at least one symptom of a condition in a subject. The term "treating" may also mean one or more of arresting, delaying the onset (i.e., the period prior to clinical manifestation of the condition) or reducing the risk of developing or worsening a condition.

[0025] The term "therapeutically effective" applied to dose or amount refers to that quantity of a compound, or exosome, stem cell, or pharmaceutical formulation that is sufficient to result in a desired clinical benefit after administration to a patient in need thereof.

[0026] As used herein, a “subject” or “patient” can be a human, non-human primate, mammal, rat, mouse, cow, horse, pig, sheep, goat, dog, cat and the like.Atty Docket No.: KIBS-001 / 01WO 357284-2006

[0027] “Mammal” includes humans and both domestic animals such as laboratory animals (e.g., mice, rats, monkeys, dogs, etc.) and household pets (e.g., cats, dogs, swine, cattle, sheep, goats, horses, rabbits), and non-domestic animals such as wildlife and the like.

[0028] All weight percentages (i.e., "% by weight” and "wt. %" and w / w) referenced herein, unless otherwise indicated, are measured relative to the total weight of the composition or pharmaceutical composition.

[0029] As used herein, ’’substantially” or ’’substantial" refers to the complete or nearly complete extent or degree of an action, characteristic, property, state, structure, item, or result. For example, an object that is "substantially" enclosed would mean that the object is either completely enclosed or nearly completely enclosed. The exact allowable degree of deviation from absolute completeness may in some cases depend on the specific context. However, generally speaking, the nearness of completion will be so as to have the same overall result as if absolute and total completion were obtained. The use of "substantially” is equally applicable when used in a negative connotation to refer to the complete or near complete lack of action, characteristic, property, state, structure, item, or result. For example, a composition that is "substantially free of' other active agents would either completely lack other active agents, or so nearly completely lack other active agents that the effect would be the same as if it completely lacked other active agents. In other words, a composition that is "substantially tree of" an ingredient or element or another active agent may still contain such an item as long as there is no measurable effect thereof.

[0030] The term “exosome” or “exosomes” means vesicles, usually small cellular membrane bound vesicles shed by most cell types, that release into the extracellular medium, and can include small, lipid-bound extracellular nanovesicles shed by cell types such as derived from multiple cell full or partial term placental tissue, umbilical tissue, or mesenchymal stem cells, and can derived naturally or synthetically, or some combination thereof.

[0031] The term “nanobubbles” means a contrast agent shown to enhance the acoustic effect of ultrasound.

[0032] As used herein, the term “pharmaceutical composition” refers to a formulation comprising at least one therapeutically active agent, which can include a cell, an extracellular vesicle, or anAtty Docket No.: KIBS-001 / 01WO 357284-2006exosome, and a pharmaceutically acceptable excipient or carrier, which may include for example a salt, buffer, or saline. A non-limiting example of pharmaceutical compositions includes tablets, capsules, solutions for i.v., gel capsules, syrup, liquid, gel, suspension, solid dispersion, or combinations thereof.Disclosure of the invention

[0033] The present invention relates to the methods described above are achieved by a) injecting an extracellular vesicle into the subject; and b) administering an ultrasound or acoustic wave to the brain of the subject. In a specific embodiment, the extracellular vesicles may be as described herein and administered to a subject in the methods described herein.Therapeutic use

[0034] The methods and compositions of the present invention relate to stimulating hair growth and / or restoring hair pigmentation in a subject. In a specific embodiment, the methods include substantially stimulating hair growth and / or restoring hair pigmentation in a subject, including a portion of the subject’s hair. In a specific embodiment, the methods may include improving or enhancing hair growth in a subject. In another specific embodiment, the methods include improving or enhancing hair pigment or hair coloration in a subject. In a specific embodiment, the hair of the subject may include white and / or gray hair wherein the coloration is reversed back to the original color of the subject, such as black, brown, or blonde hair. In a specific embodiment, the hair growth and / or restoring hair pigmentation is on the head of the subject. In another specific embodiment, the subject substantially restores original pigmentation to their hair follicles in view of the methods described herein. In a specific embodiment, the subject’s hair pigmentation is no longer white or gray. In a specific embodiment, a portion of the subject’s hair pigmentation is no longer white or gray. In a specific embodiment, at least a portion of the subject’s hair pigmentation is no longer white or gray. In a specific embodiment, the restoration of pigmentation is on the subject’s scalp or head. In a specific embodiment, at least a portion of the restoration of pigmentation is on the subject’s scalp or head. In a specific embodiment, the therapeutic use of methods above are achieved by administering the pharmaceutical compositions described herein. In a specific embodiment, the therapeutic use of methods above are achieved by a) injecting an extracellular vesicle into the subject; and b) administering an ultrasound or acoustic wave to theAtty Docket No.: KIBS-001 / 01WO 357284-2006brain of the subject. In another specific embodiment, the methods may include the prevention of baldness or the graying or whiteness of hair.

[0035] In a specific embodiment of the present invention, the extracellular vesicles are purified and isolated, which can include isolated exosomes, and are administered to a subject as described herein. In another embodiment, the methods include the step of administering a wave, such as an acoustic wave, to the brain of the subject. In view of the administration of the acoustic wave, the extracellular vesicles or exosomes may to cross over the blood brain barrier of a subject, and specifically, cross over the blood brain barrier at a higher rate or amount than if the subject was not subjected to an acoustic wave. Indeed, there are various cells or stem cells to cross over or disrupt the Blood bram barrier. For example, “Targeted Delivery of Neural Stem Cells to the Brain Using MRI-Guided Focused Ultrasound to Disrupt the Blood-Brain Barrier,” PLoS ONE 6(11): e27877.doi: 10.1371, by Burgess, et al, reports using MRI guided focused ultrasound with microbubbles to temporarily open targeted regions of the BBB to allow entry of neural stem cells in animal models. Similarly, “Cellular Mechanisms Of The Blood-Brain Barrier Opening Induced By Ultrasound In Presence Of Microbubbles,” Ultrasound in Med. & Biol., Vol. 30, No. 7, pp, 979- 989, 2004 by Sheikov, et al reports using ultrasound and microbubbles in animal models to open the BBB, but cautions tissue in the BBB can be damaged at 3W sonications. Likewise, “Noninvasive Localized Delivery Of Herceptin To The Mouse Brain By Mri-Guided Focused Ultrasound-Induced Blood-Brain Barrier Disruption,” PNAS, Vol. 103, No. 31, 11719-23 by Kinoshita, et al reports using ultrasound with microbubbles in animal models to disrupt BBB and deliver Herceptin (150 kDa) across the BBB, and notes the presence of microbubbles is required for consistent BBB opening. But it does not appear known to deliver exosomes across the BBB, to use acoustic waves or ultrasound to open the BBB in humans, or to use acoustic waves or ultrasound to safely and temporarily open the BBB in the absence of microbubbles. Accordingly, one important embodiment of the present invention is to administer a wave, such as an acoustic wave, to the brain of the subject after administration of extracellular vesicles or exosomes to said subject, such as administration by i.v.

[0036] In a specific embodiment, the acoustic wave administered to the subject is a wavelength and / or frequency that allows the extracellular vesicle, or exosome, to cross over the blood bram barrier of a subject. In a specific embodiment, the acoustic wave administered to the subject is aAtty Docket No.: KIBS-001 / 01WO 357284-2006wavelength and / or frequency that allows the extracellular vesicle, or exosome to enter the brain of a subject.

[0037] In another specific embodiment, the wave frequency may be pulsed at a frequency of 1 megahertz, (mHz), 2 mHz, 3 mHz, 4 mHz, 5 mHz, lOmHz, 100 mHz, 1Hz, 10Hz, 100Hz, 1kHz, 10kHz, 100kHz, 1MHz, 10MHz, or 100MHz, or a range therein. In another embodiment, the frequency may be in the range of about 100 khz to about 2,000 khz, about 300 khz to about 1,500 khz, about 500 khz to about 1,200 khz, about 500 khz to about 1,000 khz, or about 600 khz to about 800 khz. In another specific embodiment, the frequency of the wave administered to the subject is about 100 khz, or about 200 khz, or about 300 khz, or about 400 khz, or about 450 khz, or about 500 khz, or about 550 khz, or about 600 khz, or about 650 khz, or about 700 khz, or about 750 khz, or about 800 khz, or about 850 khz, or about 900 khz, or about 950 khz, or about 1,000 khz,

[0038] In another embodiment, the waves may be acoustic waves or ultrasonic (acoustic) waves. In a specific embodiment, the device and methods used may be as or similar to those applied in Haroon, J,, etal, SciRep 13, 17707 (2023), which is incorporated by reference in its entirely7herein.

[0039] In a specific embodiment, the acoustic waves or ultrasound system may have specific specifications. For example, the ultrasound device may comprise a transducer configured to emit about 100 khz to about 2,000 khz or about 1,000 khz, or about 750 kHz waves with a focal depth adjustable to target the brain. Accordingly, the transducer is preferably a sonic or ultrasonic transducer, but also include infrasonic and audible transducers. In another embodiment the frequency may be 650 kHz, with a use of 4 mm diameter beam width. In another embodiment, the sonic transducer may be operated at a power of at least 100 mW / cm2, 200 mW / ’cnr, more than 400 mW / cm2, more than 600 mW / cm2and more than 800 mW / cm2. In another specific embodiment, wave is at 650 kHz, and a 4 mm diameter beam width is possible, to achieve deep reach within the skull of the subject.

[0040] In another embodiment, prior to administration of the acoustic wave to the brain, the subject is subjected to magnetic resonance imaging (MRI) to determine the location of the hypothalamus or desired portion of the brain of the subject. In another specific embodiment, the imaging can be real-time imaging, e.g., MRI or ultrasound imaging, to ensure precise targeting and monitoring of acoustic energy delivery and to the intended area of the brain. In a specificAtty Docket No.: KIBS-001 / 01WO 357284-2006embodiment, the region of the subject’s brain that is subjected to the acoustic wave is selected from the group consisting of frontal lobe, parietal lobe, occipital lobe, temporal lobe, hippocampus, hypothalamus, brain stem, cerebellum amygdala, corticospinal tract, thalamus, substantia nigra, and basal ganglia and nasal submucosal injection around the inferior turbinates.

[0041] In another specific embodiment, the acoustic wave is produced by a sonic transducer or an ultrasonic transducer, or an ultrasound device.

[0042] In another specific embodiment, the acoustic wave is administered to the subject’s brain is administered between 30 minutes to 8 hrs. In another specific embodiment, the acoustic wave administered to the subject’s brain is administered between 1 hr to 4 hrs. In another specific embodiment, the acoustic wave administered to the subject’s brain is administered in the range of 1 hr to 2 hrs. The method of any one of claims 1-25, w’herein the administration to the subject is once per day, twice per day, three times per day, or 4 times per day. In another embodiment, the administration to the subject is once every month, once every 3 weeks, once every 2 weeks, once every 1 week, once every 3 days, once every 2 days, or once every day. In another embodiment, the subject is human.

[0043] In another embodiment, the administration of the acoustic wave is by Low intensity focused ultrasound (LIFU) or High-Intensity Focused Ultrasound (HIFU). In another specific embodiment, the methods further comprise the step of administering a microbubble to the patient’s blood stream. In another embodiment, the methods include the step of using the sonic transducer to sonicate the microbubble.

[0044] In a specific embodiment, the injection and / or administration of the acoustic wave is administered when the subject is asleep. In a specific embodiment, the subject is in deep or REM sleep.Extracellular Vesicles

[0045] The term “extracellular vesicles” (EVs) encompasses a group of cell-derived vesicles produced by most, if not all cell types, that are released to the extracellular environment. Different types of vesicles have been described, which differ in their properties, as well as their biogenesis. The three mam types are: apoptotic bodies (500-2000 nm), microvesicles (50—1000 nm), andAtty Docket No.: KIBS-001 / 01WO 357284-2006exosomes which are typically about 40 nm-200 nm. The extracellular vesicles may thus be a composition with a single form of isolated vesicles or an isolated combination, such as a combination of exosomes and microvesicles, including a heterogeneous mixture.

[0046] In a specific embodiment, the extracellular vesicle or exosomes are administered to the subject. These small extracellular vesicles are released by cells, carry molecular signals, and are involved in cellular communication and can cross the blood-brain barrier (BBB). After release into the extracellular space, exosomes can be internalized by recipient cells via several mechanisms including phagocytosis, micropinocytosis, endocytosis, and plasma membrane fusion. In another specific embodiment, the extracellular vesicle or exosomes are suspended in a pharmaceutical composition for administration to a subject,

[0047] In a specific embodiment, the extracellular vesicles carry miRNA, mRNA, growth factors, and immunomodulatory7proteins capable of reprogramming recipient cells in vitro and in vivo.

[0048] In a specific embodiment, the extracellular vesicles are mesenchymal stem cells MSC-derived, preferably perinatal and umbilical cord-derived MSCs based on their superior secretome and proliferative characteristics. In another embodiment, the exosomal miRNA and protein cargo are active effector molecules. In another embodiment, the exosomes or extracellular vesicles are CD63, CD81, and / or CD9 positive and the MSC lineage marker includes CD73. The present invention thus provides a method and compositions for reversing loss of hair and or pigmentation of hair in mammalian subject, comprising:(a) intravenously administering a therapeutic composition comprising isolated or purified perinatal placental MSC-derived extracellular vesicles purified by 100 kDa molecular weight cut-off (MWCO) ultrafiltration, wherein said composition comprises a heterogeneous mixture including a primary population of CD63+ / CD81+ exosomes (30- 150 nm) and a secondary population of CD9+ / CD73+ microvesicles, in a total particle dose of 1 x 109to 1 x 10’2;(b) applying transcranial Low-Intensity Focused Ultrasound (LIFU) to the hypothalamic target region, at a frequency of 250 kHz to 3 MHz, concurrently with or within 60 minutes of intravenous administration;Atty Docket No.: KIBS-001 / 01WO 357284-2006(c) wherein said LIFU induces acoustic permeabilization of the Blood-Brain Barrier at the hypothalamic target, facilitating preferential uptake of circulating extracellular vesicles into the hypothalamic parenchyma and stem cell niche;(d) resulting in observable reversal of aging phenotypes, including re-pigmentation of hair follicles previously producing gray or white hair, within approximately 3-6 months of treatment.

[0049] In a specific embodiment, the exracellular vesicles may be apoptotic bodies, microvesicles, and / or exosomes or a mixture thereof.

[0050] In a specific embodiment, extracellular vesicle or exosomes are administered to the subj ect topically, trans dermally, parenterally, intradermally, intramuscularly, intraperitoneally, intravenously, subcutaneously, or by mtranasal instillation, by intra cavitary or intravesical instillation, intraocularly, intraarterially, intralesionally, or by application to mucous membranes. In a specific embodiment, the administration is intravenously.

[0051] In a specific embodiment, the extracellular vesicle is an exosome. In a specific embodiment, the wherein the extracellular vesicle is an exosome, ranging from about 10 nm diameter to about 500 nm diameter, from about 10 nm diameter to about 500 nm diameter, from about 25 nm diameter to about 400 nm diameter, from about 40 nm diameter to about 300 nm diameter, from about 50 nm diameter to about 200 nm diameter, or from about 75 nm diameter to about 150 nm diameter.

[0052] In another specific embodiment, the extracellular vesicles or exosomes are from multiple cells, full or partial term placental tissue, umbilical tissue, or mesenchymal stem cells, and can be derived naturally or synthetically, or some combination thereof. In another embodiment, the extracellular vesicles or exosomes are from trophoblast stem cells or a combination will other cells or tissue from above. In a specific embodiment, the extracellular vesicles are from perinatal placental mesenchymal stem cells (MSCs).

[0053] In a specific embodiment, the extracellular vesicle or exosome is harvested from mesenchymal stem cells. In another specific embodiment, the mesenchymal stem cells are placental mesenchymal stem cells. In another specific embodiment, the mesenchymal stem cells are placental mesenchymal stem cells.Atty Docket No.: KIBS-001 / 01WO 357284-2006

[0054] Exosomes may be prepared by various methods known in the art, including as described in Haroon, J., etal, SciRep 13, 17707 (2023), which is incorporated by reference in its entirety herein, or in Example 1 disclosed herein. In a specific embodiment, the exosomes or extracellular vesicles are prepared by using a source cell line (such as MSCs) culture, and passing them through sterile filtration. In another specific embodiment, the sterile filtration is with a 0.22 pm sterile filtration membrane. In another embodiment, the cell line culture is isolated or purified by a centrifugal spin column. In a specific embodiment, the centrifugal spin column device with a 100 kDa MW CO membrane and centrifuged at appropriate speed and duration to drive the conditioned media through the membrane. In a specific embodiment, the centrifugal spin column filtration is after use of a sterile filtration membrane. In a specific embodiment, extracellular vesicles of 30 nm or larger are retained and or isolated from the filtration process. In a specific embodiment, intact extracellular vesicles 100 kDa or larger are retained or isolated from the filtration process. In a specific embodiment, microvesicles and / or exosomes are retained by or isolated from the filtration process.

[0055] In a specific embodiment, the filtering or purification process is performed to isolate and or purify exosome vesicles from a cell culture that are CD63+ and / or CD81+. In another embodiment, the filtering or purification process is performed to isolate and or purify or obtain retentate from a cell culture that includes microvesicles that are CD9+ and / or CD73+.

[0056] In a specific embodiment, the retained or isolated microvesicles and other extracellular vesicles are diluted or suspended in 0.9% normal saline or physiologically equivalent excipient.

[0057] The extracellular vesicle or exosome may also be in various concentrations including mean particle concentrations and protein concentrations. In a specific embodiment, the extracellular vesicle or exosome is at a concentration of about 1 x 108particles / mL to about 1x 1010particles / mL, or about 5x 108parti cles / mL to about 5x 109parti cles / mL, or about 1x 109parti cl es / mL to about 5x 109particles / mL. In another specific embodiment, the the mean particle concentration for exosomes was about 1x 109particles / mL, about 2x 109particles / mL, about 3x 109particles / mL, about 4x 109particles / mL, about 5x 109particles / mL, about 6x 109particles / mL about 7x 109parti cles / mL, about 8x 109parti cl es / mLor about 9x 109particles / mL.

[0058] In another embodiment, the extracellular vesicles may be at a certain dose or total amount for administering to a subject. In a specific embodiment, there is a range of about 1 * 108to 1 *Atty Docket No.: KIBS-001701 WO 357284-2006IO12total extracellular vesicles or particles per administered unit. In another embodiment, the range is about 1 x 108to 1x1012total extracellular vesicles or particles per administered unit. In another embodiment, the range is about 1 x 108to 1 x IO10total extracellular vesicles or particles per administered unit. In a specific embodiment, the range is about 1 x 109to about 50 x 109total extracellular vesicles or particles per administered unit; or about 2 x io9to about 30 x 109total extracellular vesicles or particles per administered unit; or about 5 x io9to about 25 x io9total extracellular vesicles or particles per administered unit; or about 10 x 109to about 20 x io9total extracellular vesicles or particles per administered unit; or about 15 x 109total extracellular vesicles or particles per administered unit.

[0059] In a specific embodiment, the extracellular vesicles are a heterogeneous mixture comprising the total extracellular vesicles or particles populations described above. In a specific embodiment, heterogeneous mixture comprises a primary population of CD63+ / CD81 + exosomes (30-150 nm) and a secondary7population of CD9+ / CD73+ microvesicles, in a total particle dose or amount ranging from about of 1 x 109to 1 x 1012or any range as described above,

[0060] In a specific embodiment, the extracellular vesicle or exosome is at a concentration of about 0,1 mg / mL to about 10 mg / mL. about 0.5 mg / mL to about 8 mg / mL, about 1 mg / mL to about 7 mg / mL, about 2 mg / mL. to about 6 mg / mL, about 2.5 mg / m. L to about 5 mg / ml,, about 3 mg / mL. to about 4mg / mL, or about 0.5 mg / mL about 1 mg / mL, about 2 mg / mL, about 3 mg / m. L, about 4 mg / mL, or about 5 mg / mL.

[0061] In another embodiment, the extracellular vesicles or exosomes may be added to a pharmaceutical composition and may be included with a pharmaceutically acceptable excipient or carrier.

[0062] In a specific embodiment, the retentate, extracellular vesicles or exosome are suspended m a suspension comprising USA and / or a salt before administration to a subject. In another specific embodiment, the salt is NaCl and the exosomes and / or extracellular vesicles (optionally including microsomes) are suspended in an about 0.8% to about 1.0% NaCl solution or a phosphate buffered saline solution. In a specific embodiment the purified or isolated extracellular vesicles, including exosomes, and / or microsomes are incorporated into a pharmaceutical composition or composition for administration to a subject. In a specific embodiment, the pharmaceutical composition or composition may include a range of about 1 x 108to l x 10i2total extracellular vesicles or particles.Atty Docket No.: KIBS-001 / 01WO 357284-2006In another embodiment, the range is about 1 * 108to 1 * 1012total extracellular vesicles or particles. In another embodiment, the range is about 1 * 108to 1 x IO10total extracellular vesicles or particles. In a specific embodiment, the range is about 1 * 109to about 50 * 109total extracellular vesicles or particles; or about 2 x io9to about 30 x 109total extracellular vesicles or particles; or about 5 x 109to about 25 x 109total extracellular vesicles or particles; or about 10 x 109to about 20 x io9total extracellular vesicles or particles; or about 15 x 109total extracellular vesicles or particles. In a specific embodiment, the amounts as provided herein are included in single composition, dose or unit dose. In a specific embodiment, the amounts described herein are for a single administration. In another embodiment, the amounts described herein are fractioned into multiple doses, including two, three, four, five, six, seven, eight, nine, or ten doses. In another embodiment, the doses and amounts described herein may be administered to a subject one time, two times, three times, four times, five times, six times, seven times, eight times, nine times, ten times or more for achieving the desired utility or result, including regrowth of hair and / or increase in pigmentation of hair in the subject,

[0063] In a specific embodiment, the compositions or pharmaceutical compositions may include more than one type of extracellular vesicle. In a specific embodiment, the composition or pharmaceutical composition may include isolated or purified exosome vesicles that are CD63+ and / or CD81+. In another embodiment, the compositions and / or pharmaceutical compositions may include isolated or purified microvesicles that are CD9 + and / or CD73+.

[0064] Perinatal placental mesenchymal stem cells (MSCs) may be used as the source cell line for preparation of extracellular vesicles Conditioned media from perinatal placental MSC cultures may be first processed using sterile filtration. Specifically, conditioned media collected from perinatal placental MSC cultures (serum-free, chemically defined) may be passed through a 0.22 pm sterile filtration membrane. The 0.22 pm-filtered conditioned media may then be loaded into a centrifugal spin column device with a 100 kDa MWCO membrane and centrifuged at appropriate speed and duration to drive the conditioned media through the membrane. The filtrate may then be discarded and the retentate — containing intact extracellular vesicles, including 30-150 nm exosomes and larger microvesicles, all with effective molecular weight as intact vesicles far exceeding 100 kDa — may be retained on the membrane and recovered. The isolated extravesiclecontaining retentate with exosomal vesicles was then resuspended in 0.9% normal saline and can then be stored and / or diluted for a specific dose of interest. The extracellular vesicles and retentateAtty Docket No.: KIBS-001 / 01WO 357284-2006may also be tested for relevant markers, including for example, CD63+ / CD81+, and also for various microvesicles, such as CD9+ / CD73+ microvesicles. In a specific embodiment, the microvesicles are CD9+ and / or CD73+ microvesicles, In another embodiment, the microvesicle population ranges in about 100-1000 nm in size. In another specific embodiment, the microvesicles are plasma membrane-derived, expressing the MSC lineage marker CD73.

[0065] In a specific embodiment, the composition or pharmaceutical composition are an isolated or purified heterogeneous mixture of extracellular vesicles. In a specific embodiment, the isolated or purified heterogeneous mixture of extracellular comprises a total amount of extracellular vesicles or particles populations in the composition or pharmaceutical composition as described above. In a specific embodiment, the heterogeneous mixture comprises a primary population of CD63+ / CD81+ exosomes (30—150 nm) and a secondary population of CD9+ / CD73+ microvesicles, in a total particle dose or amount ranging from about of 1 x IQ9to 1 x 10i2or any range as described above.

[0066] In a further embodiment of the present invention, a composition or pharmaceutical composition comprising a the extracellular vesicle or exosome and a pharmaceutically acceptable excipient or carrier is provided. In a specific embodiment, the compositions or pharmaceutically acceptable compositions may include bulk compositions or single use compositions, or single unit dose formulations as describe herein, and include a pharmaceutically acceptable carrier which may include a pharmaceutically acceptable excipient, binder, and / or diluent. In one embodiment, suitable pharmaceutically acceptable excipients include, but are not limited to, water, salt solutions, alcohol, polyethylene glycols, gelatin, lactose, amylase, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose and polyvinylpyrrolidone. Suitable formulations or pharmaceuticals for various methods of administration can be found, for example, m Remington: The Science and Practice of Pharmacy, A. Gennaro, ed., 20th edition, Lippincott, Williams & Wilkins, Philadelphia, PA.

[0067] In one embodiment, suitable pharmaceutically acceptable carriers include, but are not limited to, inert diluents and sterile aqueous or organic solutions. Pharmaceutically acceptable carriers are well known to those skilled in the art and include, but are not limited to, from about 0.01 to about 0.1 M and preferably 0.05M phosphate buffer or saline. Such pharmaceutically acceptable earners can be aqueous or non-aqueous solutions, suspensions and emulsions.Atty Docket No.: KIBS-001 / 01WO 357284-2006Examples of non-aqueous solvents suitable for use in the present application include, but are not limited to, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.

[0068] Aqueous carriers suitable for use in the present application include, but are not limited to, water, ethanol, alcoholic / aqueous solutions, glycerol, emulsions or suspensions, including saline and buffered media.

[0069] Liquid carriers suitable for use in the present application can be used in preparing solutions, suspensions, emulsions, syrups, elixirs and pressurized compounds. The active ingredient can be dissolved or suspended in a pharmaceutically acceptable liquid carrier such as water, an organic solvent, a mixture of both or pharmaceutically acceptable oils or fats.

[0070] Liquid carriers suitable for use in the present application include, but are not limited to, water (partially containing additives as above, e.g. cellulose derivatives, preferably sodium carboxymethyl cellulose solution), alcohols (including monohydric alcohols and polyhydric alcohols, e g. glycols) and their derivatives, and oils (e.g. fractionated coconut oil and arachis oil).

[0071] Liquid pharmaceutical compositions can contain emulsifying agents to disperse uniformly throughout the composition and / or combination an active ingredient or other excipient that is not soluble m the liquid carrier. Emulsifying agents that may be useful m liquid compositions and / or combinations of the present invention include, for example, gelatin, egg yolk, casein, cholesterol, acacia, tragacanth, chondrus, pectin, methyl cellulose, carbomer, cetostearyl alcohol, and cetyl alcohol.

[0072] Preservatives and chelating agents such as alcohol, sodium benzoate, butylated hydroxyl toluene, butylated hydroxyanisole, and ethylenediamine tetraacetic acid may be added at levels to improve storage stability.

[0073] A liquid composition can also contain a buffer such as guconic acid, lactic acid, citric acid or acetic acid, sodium guconate, sodium lactate, sodium citrate, or sodium acetate. Selection of excipients and the amounts used may be readily determined by the formulation scientist based upon experience and consideration of standard procedures and reference works in the field.

[0074] In one embodiment, a pharmaceutical composition is prepared for administration by injection (e.g., intravenous, subcutaneous, intramuscular, etc.). In certain of such embodiments, aAtty Docket No.: KIBS-001 / 01WO 357284-2006pharmaceutical composition comprises a carrier and is formulated in aqueous solution, such as water or physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer. In certain embodiments, other ingredients are included (e.g., ingredients that aid in solubility or serve as preservatives). In certain embodiments, injectable suspensions are prepared using appropriate liquid carriers, suspending agents and the like. Certain pharmaceutical compositions for injection are presented in unit dosage form, e.g., in ampoules, vials, or in multi-dose containers. Certain pharmaceutical compositions for injection are suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and / or dispersing agents. Certain solvents suitable for use in pharmaceutical compositions for injection include, but are not limited to, lipophilic solvents and fatty oils, such as sesame oil, synthetic faty acid esters, such as ethyl oleate or triglycerides, and liposomes. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, such suspensions may also contain suitable stabilizers or agents that increase the solubility of the pharmaceutical agents to allow for the preparation of highly concentrated solutions.

[0075] A sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, such as a solution in 1,3-butane-diol or prepared as a lyophilized powder. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile fixed oils may conventionally be employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid may likewise be used in the preparation of injectables. Formulations for intravenous administration can comprise solutions in sterile isotonic aqueous buffer. Where necessary, the formulations can also include a solubilizing agent and a local anesthetic to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together m unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampule or sachet indicating the quantity of active agent. Where the composition is administered by injection, an ampule of sterile water for injection or saline can be provided so that the ingredients can be mixed prior to administration.

[0076] Suitable formulations further include aqueous and non-aqueous sterile injection solutions that can contain antioxidants, buffers, bacteriostats, bactericidal antibiotics and solutes that renderAtty Docket No.: KIBS-001 / 01WO 357284-2006the formulation isotonic with the bodily fluids of the intended recipient; and aqueous and non¬ aqueous sterile suspensions, which can include suspending agents and thickening agents.

[0077] In other embodiments the composition of the present disclosure are administered by the intravenous route. In further embodiments, the parenteral administration may be provided in a bolus or by infusion.

[0078] In a specific embodiment, the pharmaceutical composition may comprise pharmaceutically acceptable carrier or excipients for the extracellular vesicle or exosome and may include saline and / or human serum albumin (HSA). In a specific embodiment, the pharmaceutical composition may comprise saline containing a 0.3% to 5% HSA solution, or a 0.5% to 5% HSA solution, or a 0.7% to 3% HSA solution, or a 1.0% to 3% HSA solution, or a 1.5% to 2.5% HSA solution. In another embodiment, the composition or pharmaceutical composition may include a stabilizer or additional excipients to prevent radiolysis, or aggregation of antibodies such as ascorbic acid, gentisic acid, or the like.

[0079] The pharmaceutical compositions of the present disclosure may be manufactured and / or administered in single or multiple unit dose forms.

[0080] Additional and non-limiting embodiments of the present invention are as follows:Embodiment 1: A pharmaceutical composition comprising isolated extracellular vesicle exosomes that comprise CD63+ / CD81+ surface markers and at least one pharmaceutically acceptable excipient.Embodiment 2: The pharmaceutical composition of embodiment 1, wherein the pharmaceutical composition additionally comprises isolated extracellular vesicles that are CD9+ / CD73+ microvesicles.Embodiment 3: The pharmaceutical composition of embodiment 1 or 2, wherein the exosomes and microvesicles are in a heterogeneous mixture.Embodiment 4: The pharmaceutical composition of any one of embodiments 1-3, wherein the extracellular vesicles are purified by 100 kDa MWCO ultrafiltration.Atty Docket No.: KIBS-001 / 01WO 357284-2006Embodiment 5: The pharmaceutical composition of any one of embodiments 1-4, wherein the extracellular vesicles are produced or are derived from perinatal placental MSCsEmbodiment 6: The pharmaceutical composition of any one of embodiments 1-5, wherein the composition comprises in the range of about 1 109to 1 x io12extracellular vesicle particles.Embodiment 7: The pharmaceutical composition of embodiment 6, wherein the composition comprises about 5 x io9to about 20 x 109exosomes.Embodiment 8: The pharmaceutical composition of any one of embodiments 1-7, wherein the composition is a unit dose for single administration to a subject.Embodiment 9: The pharmaceutical composition of any one of embodiments 1-8, wherein the isolated extracellular vesicles are suspended in a solution.Embodiment 10: The pharmaceutical composition of any one of embodiments 1 -9, wherein the isolated extracellular vesicles are suspended in saline or a salt solution.Embodiment 11: A method of stimulating hair growth and / or restoring hair pigmentation in a subject, comprising administering the pharmaceutical composition of any of embodiments 1-10 to a subject.Embodiment 12: The method of embodiment 11, wherein the pharmaceutical composition is administered intravenously, including by i.v.Embodiment 13: The method of embodiments 11 or 12, wherein the pharmaceutical composition is administered once, twice, three times, four times, five times, six times, seven times, eight times, nine times, or ten times to a subject.Atty Docket No.: KIBS-001 / 01WO 357284-2006Embodiment 14: The method of any one of embodiments 11-13, wherein an acoustic wave is also administered to the subject.Embodiment 15: The method of any one of embodiments 11-13, wherein an acoustic wave is also administered to the head or brain of the subject.Embodiment 16: The method of embodiment 14 or 15, wherein the acoustic wave is by a Low intensity focused ultrasound (LIFU).Embodiment 17: The method of embodiment 16, wherein said LIFU is applied at a center frequency of approximately 750 kHz, a Peak Negative Pressure (PNP) of approximately 1.5 MPa, a pulsed duty cycle of approximately 5%, and a sonication duration of 10 to 30 minutes.Embodiment 18: The method of embodiment 17, wherein LIFU targeting is guided by MRI-guided neuro-navigation, ultrasound-based skull thickness correction, or validated transcranial stereotactic coordinates.Embodiment 19: The method of any one of embodiments 14-18, wherein the acoustic wave has a frequency of between about 500 khz to about 1,000 khz.Embodiment 20: The method of any one of embodiments 11 - 19, wherein the extracellular vesicle comprises a population of exosomes, ranging from about 10 nm diameter to about 500 nm diameter.Embodiment 21: The method of embodiment 20, wherein the population of exosome has a diameter of about 40 nm to about 220 nm.Embodiment 22: The method of any one of embodiments 11-21, wherein the pharmaceutical composition is administered to the subject topically, trans dermally, parenterally, intradermally, intramuscularly, intraperitoneally, intravenously,Atty Docket No.: KIBS-001 / 01WO 357284-2006subcutaneously, or by intranasal instillation, by intracavitary or intravesical instillation, intraocularly, intraarterially, intralesionally, or by application to mucous membranes.Embodiment 23: The method of any one of embodiments 15-22, wherein the region of the subject’s brain that is subjected to the acoustic wave is selected from the group consisting of frontal lobe, parietal lobe, occipital lobe, temporal lobe, hippocampus, hypothalamus, brain stem, cerebellum amygdala, corticospinal tract, thalamus, substantia nigra, basal ganglia and nasal submucosal injection around the inferior turbinates.Embodiment 24: The method of embodiment 23, wherein the acoustic wave is specifically focused on the hypothalamus of the subject.Embodiment 25: The method of any one of embodiments 14-24, wherein prior to administration of acoustic wave to the brain, the subject is subjected to magnetic resonance imaging to determine the location of the hypothalamus of the subject.Embodiment 26: The method of any one of embodiments 14-25, wherein the acoustic wave is produced by a sonic transducer or an ultrasonic transducer, or an ultrasound device.Embodiment 27: The method of any one of embodiments 14-26, wherein the acoustic wave is administered to the subject between 30 minutes to 4 hrs.Embodiment 28: The method of embodiment 27, wherein the acoustic wave is administered is administered to the subject between 1 hr to 2 hrs.Embodiment 29: The method of embodiment 28, wherein the method further comprises the step of administering a microbubble to the subject’s blood stream.Embodiment 30: The method of embodiment 29, further comprising the step of using the sonic transducer to sonicate the microbubble.Atty Docket No.: KIBS-001 / 01WO 357284-2006Embodiment 31: The method of any one of embodiments 11-30, wherein the isolated population of extracellular vesicles, such as exosomes and / or microvesicles, enters into the brain by crossing the blood-brain barrier.Embodiment 32: The method of any one of embodiments 11-31, wherein the subject is human.Embodiment 33: The method of any one of embodiments 11-32, wherein the mean particle concentration for exosomes in the pharmaceutical composition is about 3x 109particles / mL.Embodiment 34: The method of embodiment 33, wherein the mean particle concentration for exosomes in the pharmaceutical composition is about 3x 109particles / mL,Embodiment 35: The method of embodiment 33, wherein the protein concentration of the exosome suspension m the pharmaceutical composition is about 1 mg / mL to about 6 mg / mL.Embodiment 36: The method of any one of embodiments 11-35, wherein the pharmaceutical composition is administrated to the subject once per day, twice per day, three times per day, or 4 times per day.Embodiment 37: The method of any one of embodiments 11-35, wherein the pharmaceutical composition is administrated to the subject once every month, once every 3 weeks, once every 2 weeks, once every 1 week, once every 3 days, once every 2 days, or once every day.Embodiment 37: The method of any one of embodiments 11-36, wherein after administration, the subject restores original pigmentation to at least a portion of their hair follicles.Atty Docket No.: KIBS-001 / 01WO 357284-2006Embodiment 38: The method of embodiment 37, wherein at least a portion of the subject’s hair pigmentation is no longer white or gray.Embodiment 39: The method of any one of embodiments 11 -38, wherein the restoration of pigmentation is on at least a portion of the subject’s scalp or head.EXAMPLES

[0081] Example 1: Exosome Preparation

[0082] A 5 ml vial of extravesicles are incubated with 10 pg of a biotinylated CD9 antibody (Abeam, ab28094), and 10 pg of a biotinylated CD63 antibody (Abeam, abl34331) at room temperature for 3 h with gentle rocking. After this incubation, 7.5 pg of a Streptavidin-Renilla Luciferase fusion protein (Ray Biotech, 230-00052-15) are added to the extravesicles vial, and the extravesicles are incubated for an additional hour at room temperature with gentle rocking. The extravesicles are then dialyzed in a 100 kDa cut-off dialysis tube (Thermo, 88532) at 4 °C against IX PBS for 16 h with 3 subsequent changes of PBS every 6 h.

[0083] Example 2: Treatment of Hair Restoration

[0084] Exosomes are derived from placental mesenchymal stem cells and processed as described in Example 1. The exosomes are suspended in a lx PBS solution at a concentration of 2 mg / ml, and administered to a patient by intravenous injection. The study is for 1 injection, once daily, each day for a total of 30 days for each patient, and the hair follicles on the head are examined on each day of each patient to determine hair restoration or new growth.

[0085] After each administration, real-time magnetic imaging resonance is performed on the subject to determine the location of the hypothalamus of the subject, and monitor acoustic energy delivery. Once the hypothalamus is located, an ultrasound device comprising a transducer is used, emitting 750 kHz waves to the hypothalamus of the subject for 2 hrs. This treatment demonstrates that new hair growth begins after day 10, with a noticeable statistically significant growth of new hair after 30 days of administration to each patient.Atty Docket No.: KIBS-001 / 01WO 357284-2006

[0086] Example 3: Treatment for Hair Pigment Restoration

[0087] Exosomes are derived from placental mesenchymal stem cells and processed as described in Example 1. The exosomes are suspended in a lx PBS solution at a concentration of 2 mg / ml, and administered to a patient by intravenous injection. The study is for 1 injection, once daily, each day for a total of 30 days for each patient, and the hair follicles on the head are examined on each day of each patient to determine the restoration of pigment in gray or white hair in the patient.

[0088] After each single administration, real-time magnetic imaging resonance is performed on the subject to determine the location of the hypothalamus of the subject, and monitor acoustic energy delivery'. Once the hypothalamus is located, an ultrasound device comprising a transducer is used, emitting 750 kHz waves to the hypothalamus of the subject for 2 hrs. This treatment demonstrates that new pigmentation begins after day 10, with a noticeable statistically significant change of hair color / hair pigmentation (from gray to brown or black) after 30 days of administration to each patient.

[0089] Example 4: Treatment for Hair Pigment Restoration

[0090] Preparation of exosome extravesicles

[0091] Perinatal placental Mesenchymal stem cells (MSCs) were used as the source cell line for preparation of exosome extravesicles. Conditioned media from perinatal placental MSC cultures was first processed using sterile filtration. Specifically, conditioned media collected from perinatal placental MSC cultures (serum-free, chemically defined) was passed through a 0.22 pm sterile filtration membrane. The 0,22 pm-filtered conditioned media was then loaded into a centrifugal spin column device with a 100 kDa MWCO membrane and centrifuged at appropriate speed and duration to drive the conditioned media through the membrane. The filtrate was discarded and the retentate — containing intact extracellular vesicles, including 30-150 nm exosomes and larger microvesicles, all with effective molecular weight as intact vesicles far exceeding 100 kDa — were retained on the membrane and recovered. The isolated extravesicle-containing retentate with exosomal vesicles was then resuspended in 0.9% normal saline and can then be stored and / or diluted for a specific dose of interest. The exosome vesicles and retentate were also tested forAtty Docket No.: KIBS-001 / 01WO 357284-2006relevant markers and determined to be CD63+ / CD81+. The testing of the retentate also identified CD9+ / CD73+ microvesicles.

[0092] The exosome vesicles and retentate were also tested for relevant markers The exosome extravesicle-containing retentate composition thus comprises as follows:

[0093] Primary population: CD63+ / CD81+ exosomes: 30-150 nm diameter; endosomal origin; confirmed by NTA and flow cytometry.

[0094] Secondary population: CD9+ / CD73+ microvesicles: 100-1000 nm; plasma membrane- derived; expressing the MSC lineage marker CD73. These microvesicles were thus co-retained by the 100 kDa MWCO membrane as an integral component of the composition.

[0095] Formulation: Suspended in 0.9% normal saline or physiologically equivalent excipient, sterile-filtered, endotoxin-tested, stored at 4°C or cryopreserved.

[0096] Dose: 1 * 109to 1 * 1012total extracellular vesicle particles per administered unit with approximately 15 * 109(15 billion) CD63+ / CD81+ exosomes within the total extracellular vesicle mixture.

[0097] Delivery of exosome extravesicles

[0098] One vial of exosome extravesicles containing a total of 1 * 109to 1 * 1012EV particles, including approximately 15 * 109(15 billion) CD63+ / CD81+ exosomes within a heterogeneous preparation and CD9+ / CD73+ microvesicles, suspended in 0.9% normal saline — were administered by slow intravenous infusion to the patient. No adverse events were observed during or following administration.

[0099] A LIFU Delivery System was also used to assist exosome delivery to the patient. Specifically, a focused ultrasound transducer configured to emit acoustic waves at 250 kHz to 3 MHz, capable of transcranial delivery at sufficient Peak Negative Pressure to induce stable (non-inertial) cavitation at the hypothalamic focal point.

[0100] The hypothalamic target — mediobasal hypothalamus (MBH), arcuate nucleus, paraventricular nucleus (PVN), and / or median eminence — is localized at approximately 5-7 cm from the temporal cortex in adult humans, via: (a) MRI-guided neuro-navigation with pre-acquired anatomical scan; (b) ultrasound imaging for skull thickness correction; or (c) validated transcranial stereotactic coordinates referenced to external anatomical landmarks.Atty Docket No.: KIBS-001 / 01WO 357284-2006

[0101] Transcranial LIFU was performed concurrently at time of administration of the exosome extravesicles to the patient. During IV administration, the LIFU transducer was positioned at the temporal region of the patient's skull, targeted to the hypothalamus using MRI-guided neuro¬ navigation. Operating parameters were as follows: 750 kHz center frequency, approximately 1.5 MPa PNP, approximately 5% duty cycle, 15-minute sonication (or insonation) duration. LIFU was applied concurrently to create an acoustic blood brain barrier permeabilization window coinciding with peak systemic exosomal vesicle circulation.

[0102] Approximately four months following the single administration, the patient observed unambiguous regrowth of black, pigmented hair from scalp follicles that had previously produced only white / gray hair. On a scale of 1-10, (1 being completely white and / or grey, 10 being black with full pigment), the treated hair received a score of 8, wherein before treatment was a score of 3, FIG. 1 illustrates the patient’s hair just before exosome (extravesicle composition) administration, indicating significant loss of pigmentation, i.e., white and / or gray hair on the patient’s head, and FIG. 2 illustrates 4 months after exosome vesicle administration, showing three different angles of hair growth and or increased pigmentation of hair. As documented in FIG. 2, there is systemic distribution of re-pigmented follicles across multiple scalp regions, indicating a clear therapeutic effect.

[0103] Example 5: Delivery of exosome extra vesicles to patients for hair restoration

[0104] In a 30 subject study (wherein all subjects observed white and / or gray hair on the head with an overall score of 3 or lower (1 being entirely white, 10 showing complete pigmentation of hair on the head)), one vial (5 ml composition) of exosome extravesicles containing a total of 1 * 109to 1 x 1 O'2EV particles (as isolated and prepared in Example 4), including approximately 15 x 109(15 billion) CD63+ / CD81+ exosomes within a heterogeneous preparation and CD9+ / CD73+ microvesicles, suspended in 0.9% normal saline --- are administered to said 30 subjects by slow intravenous infusion. No adverse events are observed during or following administration.

[0105] A LIFU Delivery System is also used to assist exosome delivery to 15 of the 30 subjects. Specifically, a focused ultrasound transducer configured to emit acoustic waves at 250 kHz to 3 MHz, capable of transcranial delivery at sufficient Peak Negative Pressure to induce stable (non- inertial) cavitation at the hypothalamic focal point.Atty Docket No.: KIBS-001 / 01WO 357284-2006

[0106] The hypothalamic target — mediobasal hypothalamus (MBH), arcuate nucleus, paraventricular nucleus (PVN), and / or median eminence — is localized at approximately 5-7 cm from the temporal cortex in adult humans, via: (a) MRI-guided neuro-navigation with pre-acquired anatomical scan; (b) ultrasound imaging for skull thickness correction; or (c) validated transcranial stereotactic coordinates referenced to external anatomical landmarks.

[0107] Transcranial LIFU is then performed concurrently at time of administration of the exosome extravesicles to 15 subjects (15 subjects do not receive LIFU). For the subjects receiving LIFU, the LIFU transducer is positioned at the temporal region of each subject’s skull, targeted to the hypothalamus using MRI-guided neuro-navigation. Operating parameters are as follows: 750 kHz center frequency, approximately 1.5 MPa PNP, approximately 5% duty cycle, 15-minute sonication (or insonation) duration. LIFU was applied concurrently to create an acoustic blood brain barrier permeabilization window coinciding with peak systemic exosomal vesicle circulation.

[0108] For subjects that receive LIFU, a single dose of the extracellular vesicle composition is administered and review of a therapeutic effect is scored four months later. For subjects that do not receive LIFU, three total doses of the extracellular vesicle composition are administered, once every month for three months.

[0109] Approximately four months following the first administration, 20 of the 30 subjects observe unambiguous regrowth of pigmented hair from scalp follicles that had previously produced only white / gray hair. Specifically, 12 of the 15 subjects additionally receiving LIFU observe a score of at least a 7 (on a scale of 1-10, (1 being entirely white, 10 showing complete pigmentation of hair on the head)), indicating substantial improvement in new pigmentation recovery.

[0110] Additionally, 8 of the 15 subjects not receiving LIFU observe a score of at least a 7 (on a scale of 1-10, (1 being entirely white, 10 showing complete pigmentation of hair on the head)), indicating substantial improvement in new pigmentation recovery, thus indicating that pigmentation recovery for subjects with or without receiving LIFU.

[0111] Example 6: Treatment for Hair Pigment Restoration and Hair Growth

[0112] Preparation of exosome extravesiclesAtty Docket No.: KIBS-001 / 01WO 357284-2006

[0113] Perinatal placental Mesenchymal stem cells (MSCs) were used as the source cell line for preparation of exosome extravesicles. Conditioned media from perinatal placental MSC cultures was first processed using sterile filtration. Specifically, conditioned media collected from perinatal placental MSC cultures (serum-free, chemically defined) was passed through a 0.22 pm sterile filtration membrane. The 0.22 pm-filtered conditioned media was then loaded into a centrifugal spin column device with a 100 kDa MWCO membrane and centrifuged at appropriate speed and duration to drive the conditioned media through the membrane. The filtrate was discarded and the retentate — containing intact extracellular vesicles, including 30-150 nm exosomes and larger microvesicles, all with effective molecular weight as intact vesicles far exceeding 100 kDa — were retained on the membrane and recovered. The extravesicle-containing retentate with exosomal vesicles was then resuspended in 0.9% normal saline and can then be stored and / or diluted for a specific dose of interest. The exosome vesicles and retentate were also tested for relevant markers and determined to be CD63+ / CD81+, and also containing CD9+ / CD73+ micro vesicles,

[0114] The exosome vesicles and retentate were also tested for relevant markers The exosome extravesicle-containing retentate composition thus comprises as follows:

[0115] Primary population: CD63+ / CD81+ exosomes: 30-150 nm diameter; endosomal origin; confirmed by NT A and flow cytometry.

[0116] Secondary population: CD9+ / CD73+ microvesicles: 100-1000 nm; plasma membrane-derived; expressing the MSC lineage marker CD73. These microvesicles were thus co-retained by the 100 kDa MWCO membrane as an integral component of the composition

[0117] Formulation: Suspended in 0.9% normal saline or physiologically equivalent excipient, sterile-filtered, endotoxin-tested, stored at 4°C or cryopreserved.

[0118] Dose: 1 * 109to 1 x 1012total extracellular vesicle particles per administered unit, with approximately 15 x 109(15 billion) CD63+ / CD81+ exosomes within the total extracellular vesicle mixture.

[0119] Delivery of exosome extravesicles

[0120] One vial (5 ml) of exosome extravesicles containing a total of 1 x 109to 1 x 1012EV particles, including approximately 15 x 109(15 billion) CD63+ / CD81+ exosomes within a heterogeneous preparation and CD9+ / CD73 +- microvesicles, suspended in 0.9% normal salineAtty Docket No.: KIBS-001 / 01WO 357284-2006were administered by slow intravenous infusion to each patient. A total of 24 patients received a single dose once a month for a total of three months of the exosome extravesicles and no adverse events were observed during or following administration. No concurrent LIFU was performed on these patients.

[0121] Each patient was polled, asking for any noticeable difference in hair growth or increase in hair pigmentation after exosome administration. Approximately four months following the first dose was administered, 5 of 24 patients showed a noticeable difference in hair color change on the head, showing a regrowth of pigmented hair (ex, black, blonde, brown hair), as opposed to grey and / or white hair. In addition, at least one subject noticed new and additional hair growth on the head.

[0122] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention.

[0123] While the invention has been described in connection with proposed specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth and as follows in the scope of the appended claims.

Claims

Atty Docket No.: KIBS-001 / 01WO 357284-2006WHAT IS CLAIMED IS:

1. A method of stimulating hair growth and / or restoring hair pigmentation in a subject, comprising:a) injecting an extracellular vesicle into the subject; andb) administering an acoustic wave to the brain of the subject,2. The method of claim 1, wherein the acoustic wave has a frequency of between about 500 khz to about 1,000 khz,3. The method of claim 2, wherein the extracellular vesicle is an exosome, ranging from about 10 nm diameter to about 500 nm diameter,4. The method of claim 3, wherein the exosome has a diameter of about 40 nm to about 220 nm,5. The method of any one of claims 1-4, wherein the extracellular vesicle or exosome is harvested from mesenchymal stem cells.

6. The method of claim 5, wherein the mesenchymal stem cells are placental mesenchymal stem cells.

7. The method of any one of claims 1-6, wherein the extracellular vesicle is administered to the subject topically, trans dermally, parenterally, intradermally, intramuscularly, intraperitoneally, intravenously, subcutaneously, or by intranasal instillation, by intracavitary or intravesical instillation, intra ocularly, intraarterially, intralesionally, or by application to mucous membranes.

8. The method of claim 7, wherein the administration is intravenously to the subject.Atty Docket No.: KIBS-001 / 01WO 357284-20069. The method of any one of claims 1 to 8, wherein the region of the subject’s brain that is subjected to the acoustic wave is selected from the group consisting of frontal lobe, parietal lobe, occipital lobe, temporal lobe, hippocampus, hypothalamus, brain stem, cerebellum amygdala, corticospinal tract, thalamus, substantia nigra, basal ganglia and nasal submucosal injection around the inferior turbinates.

10. The method of claim 9, wherein the acoustic wave is specifically focused on the hypothalamus of the subject.

11. The method of any one of claims 1-10, wherein prior to administration of acoustic wave to the brain, the subject is subjected to magnetic resonance imaging to determine the location of the hypothalamus of the subject.

12. The method of any one of claims 1-11, wherein the acoustic wave is produced by a sonic transducer or an ultrasonic transducer, or an ultrasound device.

13. The method of any one of claims 1-12, wherein the acoustic wave administered to the subject’s brain is administered between 30 minutes to 4 hrs.

14. The method of claim 13, wherein the acoustic wave administered to the subject’s brain is administered between 1 hr to 2 hrs.

15. The method of any one of claims 1-14, wherein the administration of the acoustic wave is by a sonic transducer, a Low intensity focused ultrasound (LIFU) or High-Intensity Focused Ultrasound (HIFU).

16. The method of any one of claims 1-15, further comprising the step of administering a microbubble to the subject’s blood stream.Atty Docket No.: KIBS-001 / 01WO 357284-200617. The method of claim 16, further comprising the step of using the sonic transducer to sonicate the microbubble.

18. The method of any one of claims 1-17, wherein the extracellular vesicle or exosome enters into the brain by crossing the blood-brain barrier.

19. The method of any one of claims 1-18, wherein the subject is human.

20. The method of any one of claims 1 -19, wherein the extracellular vesicle or exosome is suspended in a suspension comprising HSA and a salt before administration to a subject.

21. The method of claim 20, wherein the salt is NaCl and the exosomes are suspended in an about 0.8% to about 1.0% NaCl solution or PBS.

22. The method of claim 19 or 20, wherein administration is by intravenous injection of the exosome suspension in sterile 0.9% sodium chloride solution / HSA.

23. The method of any one of claims 1-22, wherein the mean particle concentration for exosomes was about 1x 109particles / mL to about 5 109particles / mL.

24. The method of claim 23, wherein the mean particle concentration for exosomes was about 3x 109particles / mL.

25. The method of any one of claims 1-24, wherein the protein concentration the exosome suspension is about 1 mg / mL to about 6 mg / mL.

26. The method of any one of claims 1-25, wherein the administration to the subject is once per day, twice per day, three times per day, or 4 times per day.Atty Docket No.: KIBS-001 / 01WO 357284-200627. The method of any one of claims 1-25, wherein the administration to the subject is once every month, once every 3 weeks, once every 2 weeks, once every’ 1 week, once every 3 days, once every 2 days, or once every day.

28. The method of any one of claims 1-27, wherein the subject restores original pigmentation to their hair follicles.

29. The method of claim 28, wherein the subject’s hair pigmentation is no longer white or gray.

30. The method of claim 28 or 29, wherein the restoration of pigmentation is on the subject’s scalp or head.

31. The method of claim 1, wherein the subject is asleep during administration of the acoustic wave.

32. The method of claim 31, wherein the subject is in deep sleep or REM sleep.

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