Compositions and methods for treating endometrial tissue

Abstract

The method for treating a subject having or at risk of having damaged endometrial tissue generally comprises administering to the subject an amount of a PEP formulation effective for treating the subject's endometrial tissue. The PEP formulation may be administered therapeutically or prophylactically. In another embodiment, the method for promoting endometrial cell proliferation generally comprises contacting endometrial cells with an amount of a PEP formulation effective for promoting endometrial cell proliferation. In another embodiment, the method for promoting wound closure by endometrial cells generally comprises administering to wounded endometrial cells an amount of a PEP formulation effective for promoting wound closure.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 257,454, filed October 19, 2021, which is incorporated by reference in its entirety. Summary of the Invention

[0002] The present disclosure, in one aspect, describes a method of treating endometrial tissue in a subject. In general, the method includes administering to the subject a purified exosome product (PEP) preparation in an amount effective to treat the endometrial tissue in the subject.

[0003] In one or more embodiments, the PEP formulation is therapeutically administered to a subject having damaged endometrial tissue. In some embodiments, the subject may have Asherman's syndrome. In other embodiments, the subject may have undergone uterine surgery.

[0004] In one or more embodiments, the PEP formulation is administered prophylactically to a subject at risk of developing endometrial tissue damage. In some of these embodiments, the PEP formulation is administered to the subject prior to uterine surgery.

[0005] In one or more embodiments, the PEP formulation is pre-administered to the subject to prepare the endometrium for fertilization.

[0006] In another aspect, the present disclosure describes a method for promoting endometrial cell proliferation. In general, the method comprises contacting endometrial cells with a PEP formulation in an amount effective to promote endometrial cell proliferation.

[0007] In another aspect, the present disclosure describes a method for promoting wound closure by endometrial cells. In general, the method comprises administering to wounded endometrial cells an effective amount of a PEP formulation to promote wound closure.

[0008] The above summary is not intended to describe each disclosed embodiment or every implementation of the present invention. The following description more particularly exemplifies illustrative embodiments. In several places throughout this application, guidance is provided through lists of examples, which examples can be used in various combinations. In each instance, the recited list serves only as a representative group and should not be interpreted as an exclusive list. [Brief description of the drawings]

[0009] [Figure 1] Confocal microscopy images of endometrial adenocarcinoma cells. PEP was labeled with DiR lipophilic dye before being added to cells in culture. After co-incubation, cells were fixed and stained with DAPI, phalloidin, or anti-CD63 fluorescent antibody. Confocal fluorescence microscopy was then performed to obtain representative images of DAPI (blue, far left), phalloidin (green, second from left), CD63 (red, center), and DiR-labeled PEP (purple, second from right), which were used to generate a merged image (far right). Scale bar is 20 μm.

[0010] [Diagram 2] Fluorescent signals of DiR and CD63 were quantified using ImageJ and compared between PEP-treated and control cells using a T-test. (A) HEC-1A cells stained with DiR. (B) HEC-1A cells stained with anti-CD63 antibody. Both CD63 and DiR were found to be statistically higher in Pep-treated cells compared to control cells.

[0011] [Diagram 3]Confocal microscopy images of human endometrial stromal cells (HESCs). PEP was labeled with DiR lipophilic dye before being added to cells in culture. After co-incubation, cells were fixed and stained with DAPI, phalloidin, or anti-CD63 fluorescent antibody. Confocal fluorescence microscopy was then performed to obtain representative images of DAPI (blue, far left), phalloidin (green, second from left), CD63 (red, center), and DiR-labeled PEP (purple, second from right), which were used to generate a merged image (far right). Scale bar is 20 μm.

[0012] [Figure 4] DiR and CD63 fluorescent signals were quantified using Image J and compared between PEP-treated and control cells using a T-test. (A) HESC stained with DiR. (B) HESC stained with anti-CD63 antibody. Both CD63 and DiR were found to be statistically higher in PEP-treated cells compared to control cells.

[0013] [Diagram 5] Representative images at three time points showing increased cell proliferation in HEC-1A cells treated with PEP exosomes versus untreated HEC-1A cells.

[0014] [Figure 6] Line graph showing proliferation of HEC-1A over 48 hours after treatment with various doses of PEP exosomes compared to untreated HEC-1A cells.

[0015] [Figure 7] Representative images at three time points showing increased cell proliferation in HESCs treated with PEP exosomes versus untreated HESCs.

[0016] [Figure 8] Line graph showing proliferation of HESCs over 48 hours after treatment with various doses of PEP exosomes compared to untreated HESCs.

[0017] [Figure 9] Representative images of HEC-1A cells following scratch wounding and treatment with serum-free medium (control) or PEP exosomes (1x1012 PEP exosomes / mL), 12 hours after scratch wounding and treatment, and 36 hours after scratch wounding and treatment.

[0018] [Figure 10] Line graph showing wound healing by HEC-1A cells treated with various doses of PEP exosomes compared to untreated HEC-1A cells.

[0019] [Figure 11] Representative images of HESCs following scratch wounding and treatment with serum-free medium (control) or PEP exosomes (1x1012 PEP exosomes / ml), 12 hours after scratch wounding and treatment, and 36 hours after scratch wounding and treatment.

[0020] [Figure 12] Line graph showing wound healing by HESCs treated with various doses of PEP exosomes compared to untreated HESCs. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] The present disclosure describes compositions and methods for treating uterine and / or cervical tissue. The compositions include purified exosome products (PEP) derived from platelet exosomes by a freeze-drying process.

[0022] The human endometrium consists of a single layer of columnar epithelium overlying a layer of connective tissue or stroma and can be divided into the upper two-thirds functional layer and the lower one-third basal layer. During a woman's reproductive life, the functional layer grows, sheds, and is repaired monthly. The endometrium has a remarkable capacity for regeneration, beginning with the lower basal layer to build a new functional layer after menstrual losses.

[0023] In Asherman's syndrome, endometrial damage disrupts the endometrial regeneration process, resulting in a scarred cavity and loss of functional endometrium. Patients may develop sequelae, including but not limited to menstrual changes, cyclical pelvic pain, and infertility. Although the etiology is not fully understood, Asherman's syndrome most commonly results from traumatic exposure to the basal layer of the myometrium itself in a state of low estrogen. The goal of Asherman's syndrome treatment is to restore an anatomically and physiologically functional cavity. The gold standard for repair of the anatomical cavity is hysteroscopic lysis of adhesions. However, there is no clear consensus on the best way to regenerate a functional endometrium and prevent the reformation of adhesions after hysteroscopic lysis. Regenerative techniques are receiving increasing attention as both a means of treatment and prevention of Asherman's syndrome.

[0024] This disclosure describes compositions comprising PEP, and therapeutic methods using the PEP compositions to treat the basal and / or functional layers of the endometrium. Compositions comprising PEP exosomes induce endometrial regeneration in two different human endometrial cell lines, HEC-1A cells and human endometrial stromal cells (HESCs). HEC-1A is a stage IA endometrial cancer cell line. HESCs are an immortalized stromal cell line with a normal karyotype that responds to hormonal stimulation. Data presented in this disclosure show that both endometrial cell lines take up PEP exosomes, the PEP composition induces cell proliferation in both cell lines, and the PEP composition increases wound closure in both cell lines. These results suggest that the PEP composition may be an effective therapeutic and / or prophylactic therapy for repairing damaged endometrial tissue and / or reducing damage to endometrial tissue.

[0025] International Patent Application No. PCT / US2018 / 065627 (published as International Publication No. WO2019 / 118817) characterizes PEP and describes a method for preparing PEP, which is incorporated herein by reference in its entirety. Briefly, PEP is a purified exosome product prepared using a freeze-drying process that produces a product with a structure different from exosomes prepared using conventional methods. For example, PEP typically has a spherical or spheroidal structure rather than a crystalline structure. Spherical or spheroidal exosome structures generally have a diameter of 300 nm or less. Typically, PEP formulations contain spherical or spheroidal exosome structures with a relatively narrow size distribution. In some formulations, PEP contains spherical or spheroidal exosome structures with a mean diameter of about 110 nm ± 90 nm, with the majority of the exosome structures having a mean diameter of 110 nm ± 50 nm, such as 110 nm ± 30 nm.

[0026] Unmodified PEP preparations, i.e., PEP preparations whose characteristics have not been altered by selection or separation of the exosome population in the preparation, show no significant differences in CD63 + Exosomes and CD63 - Naturally contains a mixture with exosomes. CD63 - Exosomes can inhibit unlimited cell proliferation, and thus CD63 + and CD63 - Unmodified PEP formulations that naturally contain exosomes can stimulate cell proliferation for wound repair and / or tissue regeneration or limit uncontrolled cell proliferation.

[0027] In addition, CD63 + By sorting exosomes, CD63 was isolated from naturally isolated PEP preparations. + Extract the exosomes and then elucidate the desired amount of CD63 + By adding back exosomes, CD63 in the PEP product - CD63 on exosomes +In one or more embodiments, the PEP formulation can be used to control the ratio of exosomes to CD63 - It may only have exosomes.

[0028] In one or more embodiments, the PEP formulation comprises CD63 + Exosomes and CD63 - Exosomes may contain both CD63 and CD63. + Exosomal CD63 - The ratio of CD63 to exosomes can vary, at least in part, depending on the amount of cell expansion desired in a particular application. + / CD63 - Exosome ratio is CD63 + Desired cell proliferation induced by exosomes and CD63 achieved through cell contact inhibition - In certain scenarios, such as tissues with non-adherent cells (e.g., blood-derived components), this ratio can be adjusted to provide the appropriate balance of cell proliferation or cell inhibition for the tissue being treated. For example, in tissues with non-adherent cells, cell-to-cell contact is not a trigger, so CD63 + The ratio of exosomes can be reduced to avoid unlimited cell proliferation. Conversely, if one wishes to expand a clonal population of cells, such as in allogeneic cell-based therapies or immunotherapy, one can use CD63 to ensure that one can obtain a large cell population from a very small source. + The ratio of exosomes can be increased.

[0029] Thus, in various embodiments, CD63 in the PEP formulation + Exosomal CD63 - The ratio to exosomes can be 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 25:1, or 30:1. In certain embodiments, the PEP product contains a 9:1 ratio of CD63 +Exosomes vs. CD63 - It is formulated to contain exosomes.

[0030] PEP uptake by cell lines Figure 1 shows confocal microscopy images of endometrial adenocarcinoma cells incubated with DiR-labeled PEP exosomes. After co-incubation, cells were fixed and stained with DAPI, phalloidin, or anti-CD63 fluorescent antibody. Control cells were stained with DAPI, phalloidin, or anti-CD63 fluorescent antibody, as HEC-1A is a human cell line and therefore the cells express CD63 as part of normal cell function. + As they contain exosomes, they stained positive for CD63. Therefore, DiR staining was used to distinguish endogenous exosomes from PEP-exosomes, which show a clear perinuclear localization. Exosomes recognized by cells are readily taken up and delivered to the perinuclear region of the cells. DiR and CD63 staining were quantified using Image J. Statistically higher amounts of both CD63 and DiR were found in PEP-treated cells compared to control cells (Figure 2).

[0031] Figure 3 shows confocal microscopy images of human endometrial stromal cells (HESCs) incubated with DiR-labeled PEP-exosomes. After co-incubation, cells were fixed and stained with DAPI, phalloidin, or anti-CD63 fluorescent antibody. Control cells again stained positive for CD63. DiR staining also shows perinuclear localization of PEP-exosomes. DiR and CD63 staining were quantified using Image J and Mann-Whitney U test with nonparametric distribution. Both CD63 and DiR were found to be statistically higher in PEP-treated cells compared to control cells (Figure 4).

[0032] Cell proliferation Figure 5 shows 10 12Microscopic images are shown demonstrating that HEC-1A cells treated with PEP exosomes / ml exhibited increased cell proliferation compared to untreated HEC-1A cells. Images were taken 12 hours after treatment and 36 hours after treatment. At both time points, increased cell proliferation and coverage of the tissue culture wells was clearly visible. Figure 6 shows that by 12 hours after treatment, all PEP doses tested, 1.25×10 11 PEP exosomes / ml ~10 12 Figure 1 shows that PEP exosomes / ml increased cell proliferation in PEP-treated HEC-1A cells compared to untreated control HEC-1A cells.

[0033] Figure 7 shows the 10 12 Microscopic images are shown demonstrating that HESCs treated with PEP exosomes / ml exhibited increased cell proliferation compared to untreated HESCs. Increased cell proliferation can be clearly seen at 12 and 36 hours post-treatment as evidenced by increased cell density in the tissue culture wells. Figure 8 shows that by 12 hours post-treatment, all PEP doses tested, 1.25 x 10 11 PEP exosomes / ml ~10 12 Figure 1 shows that PEP-exosomes / ml increased cell proliferation in PEP-treated HESCs compared to untreated control HESCs.

[0034] Effect of PEP on wound healing A scratch assay was used to model wound healing in vitro. Briefly, cells were seeded into 96-well tissue culture plates and grown until they formed a confluent monolayer. A uniform scratch was then made in the center of each well to simulate a wound. Wells were then treated with either serum-free medium (control) or serum-free medium supplemented with PEP exosomes.

[0035] Representative images of wound closure in HEC-1A cells 12 and 36 hours after treatment are shown in Figure 9. Wound closure in PEP-treated HEC-1A cells was significantly greater than that in untreated HEC-1A cells. Figure 10 shows the wound healing ability of various concentrations of PEP exosomes. After 18 hours of treatment, all concentrations of PEP exosomes induced greater wound closure in HEC-1A cells than that induced in untreated HEC-1A cells.

[0036] Similar results were observed in scratch assays using HESCs. Figure 11 shows representative images of wound closure in HESCs 12 and 36 hours after treatment. Wound closure in PEP-treated HESCs was significantly greater than in untreated HESCs. Although the difference in wound healing between PEP-treated and untreated HESCs was not as great as that observed in HEC-1A cells, all concentrations of PEP induced greater wound healing than untreated HESCs (Figure 12).

[0037] Therefore, the present disclosure describes PEP compositions and methods for treating endometrial tissue in a subject.In some cases, endometrial tissue may be damaged.Damage to endometrium includes scar tissue in endometrium and / or adhesion in endometrium.In some cases, a subject may have or be at risk of developing Asherman's syndrome, which is characterized by scar tissue and / or adhesion in uterus or cervix.

[0038] Treatment of a disease may be preventive or may be initiated after a subject shows one or more symptoms or clinical signs of the disease. A treatment that is preventive, e.g., that is initiated before a subject shows symptoms or clinical signs of the disease, is referred to herein as treatment of a subject "at risk" of having the disease. As used herein, the term "at risk" refers to a subject who may or may not actually have the risk described. Thus, for example, a subject "at risk" of having damaged endometrial tissue is a subject who has one or more risk factors associated with the disease, such as genetic predisposition, ancestry, age, or medical history (e.g., dilation and curettage (D&C), certain pregnancy complications, planned uterine surgery, etc.). Treatment can also be continued after symptoms have disappeared, e.g., to prevent or delay their recurrence.

[0039] Thus, the PEP composition can be administered before, during, or after the subject first exhibits symptoms or clinical signs of damaged endometrial tissue. By initiating treatment before the subject first exhibits symptoms or clinical signs associated with damaged endometrial tissue, the subject is less likely to experience clinical evidence of damaged endometrial tissue, the severity of symptoms and / or clinical signs of damaged endometrial tissue can be reduced, and / or the damaged endometrial tissue can be completely eliminated, compared to subjects to whom the composition is not administered. By initiating treatment after the subject first exhibits symptoms or clinical signs associated with damaged endometrial tissue, the severity of symptoms and / or clinical signs of damaged endometrial tissue can be reduced, and / or the damaged endometrial tissue can be completely eliminated, compared to subjects to whom the composition is not administered.

[0040] In one or more embodiments, the PEP composition can be administered in advance in preparation for the following event: For example, the PEP composition can be administered to prepare the uterine lining to be more receptive to the implantation of a fertilized egg. The egg can be fertilized in vitro (e.g., as a result of an in vitro fertilization (IVF) procedure) or in vivo (e.g., as a result of artificial insemination or natural insemination).

[0041] Thus, in one or more embodiments, the method comprises administering an effective amount of a PEP composition to a subject having or at risk of having damaged endometrial tissue.In this context, an "effective amount" is an amount that is effective to reduce, limit progression, improve, or eliminate, to any extent, symptoms or clinical signs associated with disease.Exemplary symptoms or clinical signs of damaged endometrial tissue include, but are not limited to, light menstrual periods (hypomenorrhea), no menstrual periods (amenorrhea), severe menstrual cramps, difficulty in conceiving, or difficulty in maintaining pregnancy.

[0042] In another embodiment, the method comprises administering to a subject in need of preparatory treatment of the endometrium an effective amount of a PEP composition, where "effective amount" in this context is an amount effective to prepare the endometrium so that it is more receptive to the implantation of a fertilized egg than the endometrium of a similar subject who has not received preparatory treatment.

[0043] Generally, the composition comprises PEP and a pharma- ceutically acceptable carrier. In a surgical setting, the PEP can be combined with a carrier suitable for delivery of the PEP formulation to endometrial tissue, such as, for example, a surgical adhesive, a tissue adhesive, and / or a support matrix (e.g., a collagen scaffold).

[0044] Thus, the method involves administering to a subject an effective amount of a PEP formulation, optionally directly to endometrial tissue. In this context, an "effective amount" is an amount effective to promote endometrial cell proliferation and / or promote wound closure in endometrial tissue.

[0045] As used herein, a "subject" can be a human or any non-human animal. Exemplary non-human animal subjects include, but are not limited to, livestock animals or companion animals. Exemplary non-human animal subjects include, but are not limited to, members of the family Hominidae (including, for example, chimpanzees, gorillas, or orangutans), subfamily Bovinae (including, for example, cattle), subfamily Caprinae (including, for example, goats), genus Ovis (including, for example, sheep), porcinae (including, for example, pigs), family Equidae (including, for example, horses), members of the family Cervidae (including, for example, deer, elk, moose, caribou, reindeer, etc.), members of the family Bisonidae (including, for example, bison), family Felidae (including, for example, domestic cats, tigers, lions, etc.), family Canidae (including, for example, domestic dogs, wolves, etc.), birds (including, for example, turkeys, chickens, ducks, geese, etc.), rodents (including, for example, mice, rats, etc.), members of the family Leporidae (including, for example, rabbits or hares), members of the family Mustelidae (including, for example, ferrets), or members of the order Chiroptera (including, for example, bats):

[0046] PEP can be formulated with a pharma- ceutically acceptable carrier to form a pharmaceutical composition. As used herein, "carrier" includes any solvent, dispersion medium, vehicle, hydrogel, coating, diluent, antibacterial and / or antifungal agent, isotonic agent, absorption delaying agent, buffer, carrier solution, suspension, colloid, and the like. The use of such media and / or agents for pharma- ceutical active substances is known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic composition is contemplated. Supplementary active ingredients can also be incorporated into the composition. As used herein, "pharma- ceutical acceptable" refers to a substance that is not biologically or otherwise undesirable, i.e., the substance can be administered to an individual together with PEP without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is included. As mentioned above, in a surgical environment, exemplary suitable carriers include surgical adhesives or tissue adhesives.

[0047] Pharmaceutical compositions containing PEP can be formulated in various forms suitable for the preferred route of administration. Thus, pharmaceutical compositions can be administered via known routes, including, for example, oral, parenteral (e.g., intradermal, transdermal, subcutaneous, intramuscular, intravenous, intraperitoneal, etc.), or topical (e.g., application to exposed nerve tissue during surgery, intranasal, intrapulmonary, intramammary, intravaginal, intrauterine, intradermal, transdermal, rectal, etc.). Pharmaceutical compositions can be administered to mucosal surfaces, such as, for example, by administration to the nasal or respiratory mucosa (e.g., by spray or aerosol). Pharmaceutical compositions can also be administered via sustained or delayed release.

[0048] Thus, the pharmaceutical composition may be provided in any suitable form, including but not limited to the form of a solution, suspension, emulsion, spray, aerosol, or any mixture.The pharmaceutical composition may be provided in a formulation that includes any pharma- ceutically acceptable excipient, carrier, or vehicle.For example, the formulation may be provided in a conventional topical dosage form, such as, for example, cream, ointment, aerosol formulation, non-aerosol spray, gel, lotion, etc.The formulation may further include one or more additives, including, for example, adjuvants, skin penetration enhancers, colorants, fragrances, flavorings, moisturizers, thickeners, etc.

[0049] The formulation may be provided in a convenient unit dosage form and can be prepared by a method known in the art of pharmacy.The method of preparing the composition containing a pharmaceutically acceptable carrier comprises the step of associating PEP with a carrier which constitutes one or more accessory ingredients.In general, the formulation can be prepared by uniformly and / or intimately associating PEP with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product into desired formulation.

[0050] The amount of PEP administered may vary depending on various factors, including but not limited to the content and / or source of PEP administered, the weight, health condition, and / or age of the subject, and / or the route of administration.Therefore, the absolute weight of PEP contained in a given unit dosage form may vary widely and depends on factors such as the species, age, weight, and health condition of the subject, and / or the method of administration.Therefore, it is not practical to generally state the amount that constitutes the amount of PEP that is effective for all possible uses.However, those skilled in the art can easily determine the appropriate amount by fully considering such factors.

[0051] In one or more embodiments, the dose of PEP can be measured in terms of PEP exosomes delivered. Thus, in one or more embodiments, the method can include administering, for example, about 1×10 6 PEP exosomes ~ approx. 1 x 10 15 This may include administering sufficient PEP to provide a dose of PEP exosomes to the subject, although in some embodiments the method may be carried out by administering PEP at a dose outside this range.

[0052] Thus, in one or more embodiments, the method comprises: 6 PEP exosomes, at least 1 × 10 7 PEP exosomes, at least 1 × 10 8 PEP exosomes, at least 1 × 10 9 PEP exosomes, at least 1 × 10 10 PEP exosomes, at least 1 × 10 11 PEP exosomes, at least 1.25 × 10 11 PEP exosomes, at least 2 × 10 11 PEP exosomes, at least 2.5 × 10 11 PEP exosomes, at least 3 × 10 11 PEP exosomes, at least 4 × 10 11 PEP exosomes, at least 5 × 10 11 PEP exosomes, at least 6 × 10 11PEP exosomes, at least 7 × 10 11 PEP exosomes, at least 7.5 × 10 11 PEP exosomes, at least 8 × 10 11 PEP exosomes, at least 9 × 10 11 PEP exosomes, at least 1 × 10 12 PEP exosomes, at least 2 × 10 12 PEP exosomes, at least 3 × 10 12 PEP exosomes, at least 4 × 10 12 PEP exosomes, at least 5 × 10 12 PEP exosomes, at least 1 × 10 13 PEP exosomes, or at least 1 × 10 14 This may include administering sufficient PEP to provide a minimum dose of PEP exosomes.

[0053] In one or more embodiments, the method comprises: 15 PEP exosomes, 1 × 10 14 PEP exosomes, 1 × 10 13 PEP exosomes, 1 × 10 12 PEP exosomes, 1 × 10 11 or 1×10 10 This may include administering sufficient PEP to provide a maximum dose of PEP exosomes of:

[0054] In one or more embodiments, the method may include administering sufficient PEP to provide a dose characterized by a range having endpoints defined by any minimum dose identified above and any maximum dose greater than the minimum dose. For example, in one or more embodiments, the method may include administering a dose of PEP of, for example, 1×10 11 ~5×10 12 Dose of PEP exosomes, 1 × 10 12 ~1×10 13 Dose of PEP exosomes, 5 × 10 12 ~1×10 13 Dose of PEP exosomes, or 1.25 × 1011 PEP exosomes ~ 1 × 10 12 The dose of PEP exosomes was 1 × 10 11 ~1×10 13 The method may include administering sufficient PEP to provide a dose of PEP exosomes. In certain embodiments, the method may include administering sufficient PEP to provide a dose equal to any minimum dose or any maximum dose listed above. Thus, for example, the method may include administering a 1×10 10 PEP exosomes, 1 × 10 11 PEP exosomes, 1.25 × 10 11 PEP exosomes, 2.5 × 10 11 PEP exosomes, 5 × 10 11 PEP exosomes, 7.5 × 10 11 PEP exosomes, 1 × 10 12 PEP exosomes, 5 × 10 12 PEP exosomes, 1 × 10 13 PEP exosomes, or 1 × 10 14 This may include administering a dose of PEP exosomes.

[0055] Alternatively, in one or more embodiments, the method may include administering sufficient PEP to provide the subject with a dose of, for example, about 0.01% to 100% solution, although in some embodiments, the method may be performed by administering PEP at a dose outside this range. As used herein, a 100% solution of PEP refers to about 75 mg of PEP solubilized in 1 ml of a liquid or gel carrier (e.g., water, phosphate buffered saline, serum-free culture medium, surgical glue, tissue adhesive, etc.). For comparison, a dose of 0.01% PEP is approximately equivalent to a standard dose of exosomes prepared using conventional methods of obtaining exosomes, such as exosome isolation from cells in vitro using standard cell conditioned media.

[0056] Thus, in one or more embodiments, the methods may include administering sufficient PEP to provide a minimum dose of at least 0.01%, at least 0.05%, at least 0.1%, at least 0.25%, at least 0.5%, at least 1.0%, at least 2.0%, at least 3.0%, at least 4.0%, at least 5.0%, at least 6.0%, at least 7.0%, at least 8.0%, at least 9.0%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, or at least 70%.

[0057] In one or more embodiments, the methods may include administering sufficient PEP to provide a maximum dose of 100% or less, 90% or less, 80% or less, 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, 20% or less, 10% or less, 9.0% or less, 8.0% or less, 7.0% or less, 6.0% or less, 5.0% or less, 4.0% or less, 3.0% or less, 2.0% or less, 1.0% or less, 0.9% or less, 0.8% or less, 0.7% or less, 0.6% or less, 0.5% or less, 0.4% or less, 0.3% or less, 0.2% or less, or 0.1% or less.

[0058] In one or more embodiments, the method may include administering sufficient PEP to provide a dose characterized by a range having endpoints defined by any minimum dose identified above and any maximum dose greater than the minimum dose. For example, in one or more embodiments, the method may include administering sufficient PEP to provide a dose between 1% and 50% of the dose, e.g., between 5% and 20% of the dose. In certain embodiments, the method may include administering sufficient PEP to provide a dose equal to any minimum dose or any maximum dose listed above. Thus, for example, the method may include administering a dose of 0.05%, 0.25%, 1.0%, 2.0%, 5.0%, 20%, 25%, 50%, 80%, or 100%.

[0059] A single dose may be administered all at once, continuously over a prescribed time, or in multiple separate doses. When multiple doses are used, each dose may be the same or different. For example, a prescribed daily dose may be administered as a single dose, continuously over a 24-hour period, or as two doses that may be equal or unequal. When multiple doses are used to deliver a single dose, the interval between doses may be the same or different. In certain embodiments, PEP may be administered by a single dose, for example, during a surgical procedure.

[0060] In certain embodiments in which multiple doses of the PEP composition are administered to the subject, the PEP composition can be administered as needed to treat the damaged endometrial tissue to the desired extent. Alternatively, the PEP composition can be administered 2, 3, 4, 5, 6, 7, 8, 9, or at least 10 times. The interval between doses can be at least one day, such as at least 3 days, at least 5 days, at least 7 days, at least 10 days, at least 14 days, or at least 21 days. The interval between doses can be at most 6 months or less, such as 3 months or less, 2 months or less, 1 month or less, 21 days or less, or 14 days or less.

[0061] In one or more embodiments, the method may include multiple administrations of PEP at intervals (in the case of two administrations) or multiple intervals (in the case of three or more administrations) characterized by a range having endpoints defined by any minimum interval and any maximum interval greater than the minimum interval identified above. For example, in one or more embodiments, the method may include multiple administrations of PEP at intervals of 1 day to 6 months, such as 3 days to 10 days. In certain embodiments, the method may include multiple administrations of PEP at intervals equal to any minimum interval or any maximum interval listed above. Thus, for example, the method may include multiple administrations of PEP at intervals of 3 days, 5 days, 7 days, 10 days, 14 days, 21 days, 1 month, 2 months, 3 months, or 6 months.

[0062] In one or more embodiments, the methods may include administering a cocktail of PEPs prepared from a variety of cell types, where each cell type has a unique profile, e.g., protein composition and / or gene expression, etc. In this manner, the PEP composition can provide a broader spectrum of endometrial tissue healing activity than if the PEP composition were prepared from a single cell type.

[0063] In the foregoing description and in the claims that follow, the term "and / or" means one or all of the listed elements or a combination of any two or more of the listed elements; the terms "comprise", "comprising" and variations thereof are to be construed as open-ended, i.e., additional elements or steps are optional and may or may not be present; unless otherwise specified, "a", "an", "the", and "at least one" are used interchangeably to mean one or more; and the recitation of numerical ranges by endpoints includes all numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.).

[0064] In the foregoing description, certain embodiments may be described alone for clarity. Throughout this specification, references to "one embodiment," "embodiment," "particular embodiment," "some embodiments," or the like, mean that the particular features, configurations, compositions, or characteristics described in connection with that embodiment are included in at least one embodiment of the present disclosure. Thus, the appearance of such phrases in various places throughout this specification does not necessarily refer to the same embodiment of the present disclosure. Furthermore, certain features, configurations, compositions, and characteristics may be combined in any suitable manner in one or more embodiments. Furthermore, certain features, configurations, compositions, and characteristics may be combined in any suitable manner in one or more embodiments. Thus, features described in the context of one embodiment may be combined with features described in the context of a different embodiment, unless the features are necessarily mutually exclusive.

[0065] In any method disclosed herein that includes separate steps, the steps may be performed in any practicable order, and, if desired, any combination of two or more steps may be performed simultaneously.

[0066] As used herein, the terms "preferred" and "preferably" refer to embodiments of the invention that may provide certain benefits, under particular circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, nor is it intended to exclude other embodiments from the scope of the invention.

[0067] The present invention is illustrated in the following examples, it being understood that the specific examples, materials, amounts and procedures are to be interpreted broadly in accordance with the scope and spirit of the invention described herein.

[0068] Exemplary embodiments Embodiment 1 is a method of treating endometrial tissue in a subject, comprising administering to the subject a PEP formulation in an amount effective to treat the endometrial tissue in the subject.

[0069] Embodiment 2 is the method of embodiment 1, wherein the subject has damaged endometrial tissue.

[0070] Embodiment 3 is the method of any one of Embodiments 1 or 2, wherein the subject has Asherman's syndrome.

[0071] Embodiment 4 is the method of any one of embodiments 1 to 3, wherein the subject has had uterine surgery.

[0072]

[0023] Embodiment 5 is the method of any one of embodiments 1-4, wherein the subject is at risk of having damaged endometrial tissue.

[0073] Embodiment 6 is the method of any one of embodiments 1 to 5, wherein the PEP formulation is administered to the subject prior to uterine surgery.

[0074] Embodiment 7 is a method according to any one of embodiments 1 to 6, wherein the PEP formulation is administered in preparation for a treatment.

[0075] Embodiment 8 is the method of any one of embodiments 1 to 7, wherein the PEP formulation is administered prior to the in vitro fertilization procedure.

[0076] Embodiment 9 is a method of promoting endometrial cell proliferation, comprising contacting endometrial cells with a PEP formulation in an amount effective to promote endometrial cell proliferation.

[0077] Embodiment 10 is a method of promoting wound closure by endometrial cells, comprising administering to wounded endometrial cells a PEP formulation in an amount effective to promote wound closure.

[0078] Embodiment 11 is the method according to any one of embodiments 1 to 10, wherein the PEP formulation comprises CD63+ exosomes.

[0079] Embodiment 12 is a method for preparing a PEP formulation comprising administering to a patient a dose of at least 1×10 9 The method according to any one of embodiments 1 to 11, having a concentration of PEP exosomes / mL.

[0080] In embodiment 13, the PEP formulation is 1×10 9 ~1×10 11 The method according to any one of embodiments 1 to 11, having a concentration of PEP exosomes / mL.

[0081] Embodiment 14 is a method according to any one of embodiments 10 to 13, wherein the wound closes twice as fast as a comparable wound not treated with the PEP formulation.

[0082] Embodiment 15 is the method of any one of embodiments 9 or 11-13, wherein the cells proliferate at least twice as fast as comparison cells not treated with the PEP formulation.

[0083] Embodiment 16 is a method according to any one of embodiments 1 to 8, wherein the treatment prevents the formation of adhesions.

[0084] Embodiment 17 is a method according to any one of embodiments 1 to 8, wherein the treatment restores uterine functionality. EXAMPLES

[0085] DiR labeling of PEP Lyophilized PEP was prepared as previously described (WO 2019 / 118817) and then reconstituted in heparinized serum-free medium and incubated with a lipophilic dye (DiR) to label exosomes. The formulation was then filtered, washed with phosphate-buffered saline (PBS) and administered to cells in culture. Approximately 30,000 HEC-1A cells or HESCs were seeded on both sides of a dual-chamber glass slide. PEP was reconstituted in 4.6 mL serum-free medium + 400 μl heparin. Exosomes were labeled with the lipophilic membrane-specific far-red dye DiR (1,1'-dioctadecyl-3,3,3',3'-tetramethylindotricarbocyanine iodide). 1 mL of reconstituted PEP was added to one side of the dual-chamber slide. 1 mL of serum-free medium without PEP was added to the other side and the slides were incubated for 1 hour, then rinsed and fixed overnight in paraformaldehyde (PFA). After fixation with PFA, cells were stained with anti-CD63 antibody, phalloidin, and DAPI. Anti-CD63 antibody was visualized with a secondary antibody labeled with ALEXA FLUOR 555. Phalloidin was used to visualize actin. DAPI was used to visualize nuclei. Coverslips were mounted with Prolonged gold anti-fade reagent with DAPI. Images of each label were obtained by confocal fluorescence microscopy and are shown in Figures 1 and 3.

[0086] Both HESC and HEC-1A cells treated with PEP showed punctate cytosolic localization of DiR, indicating that PEP was taken up by the cells and localized to discrete perinuclear locations (Figure 1, Figure 3). This observation is consistent with the knowledge that endocytic vesicles containing PEP are recognized and localized by cells upon endocytosis. HEC-1A cells not treated with PEP showed low levels of CD63 staining, consistent with the knowledge that HEC-1A inherently harbor exosomes that stain with CD63. HESC showed minimal levels of CD63 staining.

[0087] The image data from Figures 1 and 3 are quantified in Figures 2 and 4. Statistically significant differences in the amounts of DiR and CD63 between treated and untreated cells were observed in both HEC-1A cells (Figure 2) and HESCs (Figure 4).

[0088] Cell proliferation assay To examine cell proliferation, cells were imaged and counted throughout treatment with PEP-exosomes using an INCUCYTE S3 imaging system (Sartorius AG, Goettingen, Germany).

[0089] To perform the assay, cells were thawed from cryopreservation and subsequently seeded in 96-well tissue culture plates in standard growth medium. HEC-1A cells were seeded at approximately 5,000 cells / well. HESCs were seeded at approximately 1,000 cells / well. Seeded cells were rinsed once with PBS. 50 μl of serum-free medium was added to control wells. Experimental wells were rinsed with 50 μl of 1.25 × 10 11 PEP exosomes / ml, 2.5 x 10 in 50 μl 11 PEP exosomes / ml, 5 x 10 in 50 μl 11 PEP exosomes / ml, 7.5 x 10 in 50 μl 11 PEP exosomes / ml or 1 x 10 12Plates were then incubated for 48 h in a live cell analysis system (INCUCYTE, Sartorius AG, Goettingen, Germany).

[0090] Images of cells in each well were acquired at regular intervals throughout the incubation period using an imaging system (INCUCYTE C3, Sartorius AG, Gottingen, Germany). After the incubation period, images were analyzed using the accompanying software package (INCUCYTE, Sartorius AG, Gottingen, Germany) to assess the cell confluency in each well.

[0091] Control untreated HEC-1A cells and 50 μl of 1 x 10 12 Exosomes / mL (5 × 10 for total exosomes) 10 Representative images of HEC-1A cells treated with PEP exosomes are shown in Figure 5. HEC-1A cells treated with PEP exosomes proliferated faster than control cells. Figure 6 shows quantification data from multiple images of HEC-1A cells treated with various concentrations of PEP exosomes. Cells treated with higher concentrations of PEP proliferated faster.

[0092] 50 μl of untreated hESCs for control and 1 x 10 12 Exosomes / mL (5 × 10 for total exosomes) 10 Representative images showing HESCs treated with PEP exosomes are shown in Figure 7. Similar to HEC-1A cells, HESCs treated with PEP exosomes proliferated faster than control cells. Figure 8 shows quantified data from multiple images of HESCs treated with each concentration of PEP exosomes. Strikingly, HESCs treated with the highest concentration of PEP exosomes showed nearly 400% proliferation compared to control cells, which showed less than 50% proliferation at 48 hours. HESCs treated with the three highest concentrations of PEP showed statistically significantly increased levels of proliferation just 6 hours after treatment.

[0093] Scratch assay (in vitro wound healing) Cells were seeded into 96-well plates using a multichannel pipette and allowed to attach and grow to confluency: HEC-1A cells were seeded at a confluency of 50,000 cells / well; HESCs were seeded at a confluency of 30,000 cells / well.

[0094] When cells were confluent, a uniform scratch wound was created in the center of each well using a wounding tool (Sartorius AG, Goettingen, Germany) to disrupt the monolayer. After confirming a uniform scratch by microscopy, the wells were rinsed once with PBS and either serum-free medium or medium containing various concentrations of PEP was added as described for the cell proliferation assay. The plates were then placed in a live cell analysis system (INCUCYTE, Sartorius AG, Goettingen, Germany) housed in a tissue culture incubator for 48 hours.

[0095] Images of each well were captured and analyzed as described above for the cell proliferation assay.

[0096] Control untreated HEC-1A cells and 50 μl of 1 x 10 12 Exosomes / mL (5 × 10 for total exosomes) 10 Representative images showing wound healing of HEC-1A cells treated with 1 × 10 exosomes are shown in Figure 9. After 36 hours, HEC-1A cells treated with PEP exosomes had proliferated to nearly cover the wound. In contrast, untreated HEC-1A cells had proliferated to only partially cover the wound at 36 hours. Figure 10 shows quantification of the image data of HEC-1A cells treated with various concentrations of PEP exosomes. Cells treated with PEP exosomes showed more wound healing as measured by wound density. This effect was overall dose-dependent. Six hours after treatment, 50 μl of 1 × 10 exosomes were administered to 1 × 10 exosomes. 12 5 x 10 exosomes / mL and 50 µl 11Cells treated with exosomes / mL had statistically significant wound density compared to untreated HEC-1A cells. After 18 hours of treatment, cells treated with each concentration of PEP exosomes had statistically significant wound density compared to untreated HEC-1A cells.

[0097] 50 μl of untreated hESCs for control and 1 x 10 12 Exosomes / mL (5 × 10 for total exosomes) 10 Representative images showing wound healing of HESCs treated with 1 × 10 exosomes are shown in Figure 11. After 36 hours, HESCs treated with PEP exosomes had proliferated to cover the wound. After 36 hours, control untreated HESCs had proliferated to partially but not completely cover the wound. Figure 12 shows quantification of the image data of HESCs treated with each concentration of PEP exosomes. Interestingly, this effect did not appear to be dose-dependent; rather, cells treated with all concentrations of PEP exosomes showed increased wound density compared to untreated HESCs. After 36 hours, 50 μl of 1 × 10 exosomes increased wound density compared to untreated HESCs. 12 7.5 x 10 exosomes / mL in 50 µl 11 Exosomes / mL, or 1.25 x 10 in 50 μl 11 HESCs treated with exosomes / mL exhibited statistically significantly higher wound density than control HESCs. These results suggest an unexpected relationship between the dose of PEP-exosomes administered and wound healing in HESCs.

[0098] The complete disclosures of all patents, patent applications, and publications cited herein, as well as electronically available materials (including, for example, nucleotide sequence deposits in GenBank and RefSeq, and amino acid sequence deposits in SwissProt, PIR, PRF, PDB, and translations from annotated coding regions in GenBank and RefSeq) are incorporated by reference in their entirety. In the event of any inconsistency between the disclosure of this application and the disclosure of any document incorporated herein by reference, the disclosure of this application shall control. The foregoing detailed description and examples are given for clarity of understanding only. They should not be understood as being unnecessarily limiting. The invention is not limited to the exact details shown and described, and modifications obvious to one skilled in the art will be included within the scope of the invention as defined by the claims.

[0099] Unless otherwise indicated, all numbers expressing quantities of ingredients, molecular weights, and the like used in the specification and claims should be understood to be modified in all instances by the term "about." Accordingly, unless otherwise indicated, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by ordinary rounding techniques.

[0100] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values ​​set forth in the specific examples are reported as precisely as possible, however, all numerical values ​​inherently contain ranges necessarily resulting from the standard deviation found in their respective testing measurements. All headings are for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading, unless so specified.

Claims

1. 1. A pharmaceutical composition comprising a purified exosome product (PEP) preparation for use in a method for treating endometrial tissue in a subject, the method comprising administering to the subject an amount of the PEP preparation effective to treat the endometrial tissue of the subject.

2. The pharmaceutical composition of claim 1 , wherein the subject has damaged endometrial tissue.

3. 10. The pharmaceutical composition of claim 1, wherein the subject has Asherman's syndrome.

4. The pharmaceutical composition of claim 2, wherein the subject has undergone uterine surgery.

5. The pharmaceutical composition of claim 1 , wherein the subject is at risk of having damaged endometrial tissue.

6. 6. The pharmaceutical composition of claim 5, wherein the PEP formulation is administered to the subject prior to uterine surgery.

7. The pharmaceutical composition of claim 1 , wherein the PEP formulation is administered in preparation for a treatment.

8. 8. The pharmaceutical composition of claim 7, wherein the PEP formulation is administered prior to an in vitro fertilization procedure.

9. 1. A pharmaceutical composition comprising a purified exosome product (PEP) preparation for use in a method for promoting proliferation of endometrial cells, the method comprising contacting the endometrial cells with an amount of the PEP preparation effective to promote proliferation of the endometrial cells.

10. 1. A pharmaceutical composition comprising a purified exosome product (PEP) preparation for use in a method for promoting wound closure by endometrial cells, the method comprising administering to wounded endometrial cells an amount of the PEP preparation effective to promote wound closure.

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