A micronized composition for wound healing prepared from intact human amniotic-chorionic tissue having an intact middle spongy layer therebetween

Abstract

A sterile micronized composition that is configured for wound healing and is prepared from intact human placental tissue. The sterile micronized composition comprises micronized human amnion, micronized human chorion, and micronized human intermediate spongiosa layer. In certain embodiments, the composition is prepared from intact placental tissue comprising a human amnion layer, a human chorion layer, and an intermediate spongiosa layer located therebetween, wherein the human amnion layer and the human chorion layer are not separated from each other immediately prior to micronization.

Description

Technical Field

[0001] The present invention generally relates to the field of compositions prepared from human placental tissue, and more particularly to a micronized composition or a sterile micronized composition prepared from intact human placental tissue (human amnion, human chorion, and human intermediate spongiosa are connected to each other and the cross-section is intact) for healing the wounds of a subject to be treated.

Background Art

[0002] The human placenta is an organ that develops in the uterus immediately after the implantation (embryo formation) of a human fertilized egg and attaches the human fertilized egg to the uterine wall. The human placenta is composed of soft tissue, chorion, amnion, and an umbilical cord connecting to the fetus. During the development process of the fetus, the amnion and chorion fuse to form a chorion-amnion membrane that wraps the amniotic fluid and the fetus. The overall thickness of the fetal membrane (amnion sac) is about 0.25 mm at term. This includes the innermost amnion layer and the outermost chorion layer.

[0003] In the uterus, the amnion is in direct contact with the amniotic fluid. The amnion has three layers, which contain high concentrations of proteoglycans and glycoproteins together with type I and types III-VII collagens, fibronectin, and laminin. The functions of the amnion membrane include physical protection of the fetus, protection from bacterial infection, pH regulation, and secretion of growth factors and other molecules. These perform antibacterial and anti-inflammatory functions.

[0004] The chorion is the outer layer of the human placenta that contacts maternal cells. The chorion forms caudal - like structures called chorionic villi, which supply blood and nutrients from the mother to the fetus. The amnion and chorion are separated by a jelly - like matrix (chorionic fluid) until these two membranes "fuse" at 11 - 13 weeks after pregnancy. The chorion is 3 - 4 times thicker than the amnion and has three layers: an intermediate / reticular layer (adjacent to the amnion), a basement membrane, and a trophoblast layer. The reticular layer contains type I, III, IV, V, and VI collagen. The basement membrane contains, in addition to type IV collagen, fibronectin and laminin.

[0005] In 1910, John Davis first reported using the amniotic membrane (AM or amnion) as a surgical material in skin grafting. Davis showed that in skin grafting, the amniotic membrane produced better results than xenografts or cadaveric dressings (6). Since then, the human amniotic membrane has been used in regenerative medicine due to its good biological and mechanical properties. It has benefited from these properties in numerous applications. For example, amniotic membranes for the management and treatment of skin burns have been used in over 200 clinical trials. Similarly, amniotic membranes have shown potentially beneficial results when used for chronic wounds, urinary problems, dental and oral treatments, ophthalmic indications, and orthopedic treatments.

[0006] The human chorion itself is generally not used in regenerative medicine, but the combination of the amnion and chorion (AC) has been used in the field. Similar to AM, AC has also been used in numerous applications in various forms.

[0007] The main use of a composition containing both human amnion and human chorion is wound healing. However, such compositions tend to become highly processed laminates that have lost various endogenous growth factors found in the human placenta in vivo due to extensive processing. Further, these compositions have a reduced and / or limited wound healing ability because various endogenous growth factors are lost during processing. For example, U.S. Patent No. 8,357,403, U.S. Patent No. 8,372,437, U.S. Patent No. 8,409,626, and U.S. Patent Application No. 17 / 097,350 each disclose various formulations in which the amnion is physically separated from the chorion and the portion of the intermediate sponge layer therebetween is partially lost and / or intentionally completely removed during separation. Further, in each of U.S. Patent No. 8,357,403, U.S. Patent No. 8,372,437, U.S. Patent No. 8,409,626, and U.S. Patent Application No. 17 / 097,350, the separated amnion and / or separated chorion are each further processed into a laminate or are further individually micronized with a substantial portion of the intermediate sponge layer at least partially and / or completely removed as compared to the human placenta in vivo. Thus, the compositions disclosed in U.S. Patent No. 8,357,403, U.S. Patent No. 8,372,437, U.S. Patent No. 8,409,626, and U.S. Patent Application No. 17 / 097,350, and more particularly, the growth factor profiles of these compositions, are significantly different from the growth factor profile of the human placenta in vivo, and the wound healing ability of these compositions is reduced and / or limited due to the loss of various endogenous growth factors during processing of these compositions.

[0008] In addition to the above, various placenta-based compositions in the field of regenerative medicine utilize harsh processing techniques such as cryo-fracture (summarized in Taylor, J. (2008), Cryo-Fracture or Freeze-Fracture, a Method to Expose Internal Tissue Surfaces and Cell Surfaces for Viewing in the Scanning Electron Microscope, Microscopy Today, 16(4), 56-59, doi:10.1017 / S1551929500059812) and / or lyophilization (summarized in https: / / www.fda.gov / inspections-compliance-enforcement-and-criminal-investigations / inspection-guides / lyophilization-parenteral-793, accessed on June 27, 2022). Therefore, during the processing of these compositions, various endogenous growth factors are lost compared to human placenta tissue in vivo. For example, U.S. Patent No. 6,933,326 discloses the treatment of human amnion and chorion using a cryo-fracture process. To perform cryo-fracture, water must be present within the specific cells and / or tissue in which this process is carried out. In this process, the cells and / or tissue are rapidly and abruptly cooled (i.e., frozen) and then fractured under vacuum. The water present within the cells and / or tissue forms ice crystals upon rapid cooling, disrupting the cell membranes of the cells and / or tissue in which cryo-fracture is being performed. The presence of water prior to cryo-destruction of the cells / tissue increases the likelihood of various degradation processes (such as apoptosis, necroptosis, lysosomal degradation, etc.) occurring within the cells / tissue, further leading to a decrease in endogenous growth factors found in human placenta tissue and further reducing and / or limiting the wound healing ability of the composition.Similarly, due to the physical process in which cells / tissues are destroyed by ice crystals during freeze fragmentation, the likelihood of a degradation process (lysosomal degradation) occurring within the cells / tissues increases, further leading to a decrease in endogenous growth factors seen in human placental tissue in vivo. During the processing of the composition, various endogenous growth factors decrease, resulting in a further reduction and / or limitation of the wound healing ability of the composition formed by freeze fragmentation and / or lyophilization. SUMMARY OF THE INVENTION

[0009] From the above, an object of the present invention is to provide a micronized composition or a sterile micronized composition comprising human amnion, human chorion, and an intermediate sponge layer, and containing a growth factor profile that includes and / or is similar to the growth factor profile of the human placenta in vivo. These compositions are preferably minimally processed and, furthermore, exhibit better wound healing properties more advantageously because various endogenous growth factors are maintained. In a specific embodiment, the compositions disclosed herein comprise an intact placental tissue (intact cross-section) comprising a human amnion layer, a human chorion layer, and an intact human intermediate sponge layer located between and connecting the human amnion layer and the human chorion layer, and are micronized compositions and / or sterile micronized compositions prepared therefrom. By not separating the human amnion layer and the human chorion layer from each other immediately prior to micronization, various endogenous growth factors are maintained in the composition as compared to the growth factor profile of the human placenta in vivo. Furthermore, the compositions disclosed herein advantageously are not subjected to harsh processing conditions such as freeze fragmentation and / or lyophilization during the preparation of the disclosed compositions, thus avoiding the degradation of growth factors commonly seen in freeze fragmentation and / or lyophilization.

[0010] In certain embodiments, the present specification discloses a micronized composition or a sterile micronized composition that is configured for wound healing and prepared from intact human placental tissue, and the micronized composition or the sterile micronized composition comprises (a) micronized human amnion, (b) micronized human chorion, and (c) micronized human intermediate sponge layer. In certain embodiments, the present specification discloses a micronized composition or a sterile micronized composition that is configured for wound healing and prepared from intact human placental tissue, and the micronized composition or the sterile micronized composition consists essentially of (a) micronized human amnion, (b) micronized human chorion, and (c) micronized human intermediate sponge layer. In certain embodiments, the present specification discloses a micronized composition or a sterile micronized composition that is configured for wound healing and prepared from intact human placental tissue, and the micronized composition or the sterile micronized composition consists of (a) micronized human amnion, (b) micronized human chorion, and (c) micronized human intermediate sponge layer.

[0011] In certain embodiments, the micronized human amnion constitutes 10% - 20% of the total weight of the micronized composition or the sterile micronized composition.

[0012] In certain embodiments, the micronized human chorion constitutes 65% - 75% of the total weight of the micronized composition or the sterile micronized composition.

[0013] In certain embodiments, the micronized human intermediate sponge layer constitutes 10% - 20% of the total weight of the micronized composition or the sterile micronized composition.

[0014] In certain embodiments, the micronized composition or the sterile micronized composition contains 1.0×10 2 ~5.0×10 4 pg / cm 2 of interleukin-1 receptor antagonist (IL-1ra), 5.0×10 2 ~1.0×10 4 pg / cm 2 of hepatocyte growth factor (HGF), and 5.0×10 2 ~5.0×10 3 pg / cm 2of vascular endothelial growth factor receptor 1 (VEGFR1) and 1.0×10 7 ~1.0×10 8 pg / cm 2 of hyaluronic acid (HA), and at least two of glycosaminoglycan (GAG) and collagen.

[0015] In certain embodiments, the micronized composition or the sterile micronized composition is 1.0×10 2 ~5.0×10 4 pg / cm 2 of IL-1ra, 5.0×10 2 ~1.0×10 4 pg / cm 2 of HGF, 5.0×10 2 ~5.0×10 3 pg / cm 2 of VEGFR1, 1.0×10 7 ~1.0×10 8 pg / cm 2 of HA, and at least three of GAG and collagen.

[0016] In certain embodiments, the micronized composition or the sterile micronized composition is 1.0×10 2 ~5.0×10 4 pg / cm 2 of IL-1ra, 5.0×10 2 ~1.0×10 4 pg / cm 2 of HGF, 5.0×10 2 ~5.0×10 3 pg / cm 2 of VEGFR1, 1.0×10 7 ~1.0×10 8 pg / cm 2 of HA, and at least four of GAG and collagen.

[0017] In certain embodiments, the micronized composition or the sterile micronized composition is 1.0×10 2 ~5.0×10 4 pg / cm 2 of IL-1ra, 5.0×10 2 ~1.0×104 pg / cm 2 of HGF, and 5.0×10 2 ~5.0×10 3 pg / cm 2 of VEGFR1, and 1.0×10 7 ~1.0×10 8 pg / cm 2 of HA, GAG, and collagen, and contains at least 5 of them.

[0018] In certain embodiments, the micronized composition or the sterile micronized composition is 1.0×10 2 ~5.0×10 4 pg / cm 2 of IL-1ra, 5.0×10 2 ~1.0×10 4 pg / cm 2 of HGF, 5.0×10 2 ~5.0×10 3 pg / cm 2 of VEGFR1, and 1.0×10 7 ~1.0×10 8 pg / cm 2 of HA, GAG, and collagen, and contains each of them.

[0019] In certain embodiments, the micronized human amnion has a diameter of 1 μm to 500 μm, more preferably 10 μm to 400 μm, and most preferably 10 μm to 300 μm.

[0020] In certain embodiments, the micronized human chorion has a diameter of 1 μm to 500 μm, more preferably 10 μm to 400 μm, and most preferably 10 μm to 300 μm.

[0021] In certain embodiments, the micronized human intermediate sponge layer has a diameter of 1 μm to 500 μm, more preferably 10 μm to 400 μm, and most preferably 10 μm to 300 μm.

[0022] In certain embodiments, the micronized composition and / or the sterile micronized composition is not cryogenically milled.

[0023] In certain embodiments, the micronized composition or sterile micronized composition comprises the human amnion layer, the human chorion layer, and the intact human spongy intermediate layer located between and connecting the human amnion layer and the human chorion layer, and is prepared from intact placental tissue (with an intact cross-section), and immediately prior to micronization, the human amnion layer and the human chorion layer are not separated from each other.

[0024] In certain embodiments, the micronized composition or sterile micronized composition is sterilized, dehydrated, and standardized to the weight of the placental tissue.

[0025] In certain embodiments, the micronized composition or sterile micronized composition is standardized by the following formula.

Number

[0026] In certain embodiments, the micronized composition or sterile micronized composition is configured for wound packing, treatment of wounds with irregular surfaces and / or surrounding boundaries, exudative wounds, dental wounds and / or oral wounds, or any combination thereof.

[0027] In certain embodiments, a pharmaceutical product comprising the micronized composition or sterile micronized composition is also disclosed.

[0028] In certain embodiments, a therapeutic aerosol configured to be administered intranasally and / or by pulmonary administration to a subject in need of treatment and comprising the micronized composition or sterile micronized composition is also disclosed. The therapeutic aerosol is configured to treat lung disorders, tracheal disorders, and combinations thereof in a subject in need of treatment.

[0029] In certain embodiments, a therapeutic dressing configured for the topical wound treatment of a subject in need thereof and comprising the disclosed micronized composition or sterile micronized composition is also disclosed. The therapeutic dressing comprises a cellulose matrix, a polymer matrix, or a combination thereof, and the disclosed micronized composition or sterile micronized composition is incorporated therein. The cellulose matrix comprises a carboxyalkyl cellulose matrix or a hydroxyalkyl cellulose matrix. In certain embodiments, the therapeutic dressing is configured to sustainably release the sterile micronized composition to the wound over a predetermined period (e.g., 1 day to 7 days, more preferably 2 days to 3 days).

[0030] In certain embodiments, the compositions disclosed herein do not use placental discs and / or are not formed into placental disc shapes after the production of the disclosed micronized compositions and / or sterile micronized compositions. In certain embodiments, the disclosed micronized compositions and / or sterile micronized compositions do not further utilize carriers (e.g., liposomes, polymeric micelles, microspheres, nanoparticles, and emulsions). In certain embodiments, the amnion and chorion of the micronized composition and sterile micronized composition are not separated immediately prior to micronization and do not include recombining or re-laminating the amnion, chorion, and / or intermediate spongiosa layer immediately prior to micronization. In certain embodiments, the disclosed micronized compositions and / or sterile micronized compositions are not injectable compositions (e.g., subcutaneous injectable compositions, intravenous injectable compositions, and / or intramuscular injectable compositions) and do not further contain anesthetics (e.g., lidocaine, bupivacaine, mepivacaine, etc.).

[0031] Embodiments of the present invention may include any one or a combination of the above features and configurations. Additional features, aspects, and advantages of the present invention are described in the following detailed description, and in part will be readily apparent to those skilled in the art from that description, or will be recognized by practicing the present invention as described herein. It is to be understood that both the foregoing general description and the following detailed description present various embodiments of the present invention, and are intended to provide an overview or framework for understanding the nature and characteristics of the claimed invention. The accompanying drawings are included to provide a further understanding of the present invention, and are incorporated herein and constitute a part of this specification.

Brief Description of the Drawings

[0032] These, as well as other features, aspects, and advantages of the present invention, will be better understood by reading the following detailed description of the invention with reference to the accompanying drawings.

[0033]

Figure 1

[0034]

Figure 2

[0035]

Figure 3

[0036]

Figure 4

[0037]

Figure 5

Best Mode for Carrying Out the Invention

[0038] Hereinafter, the present invention will be described in more detail with reference to the accompanying drawings showing exemplary embodiments of the present invention. However, the present invention can be embodied in many different forms and should not be construed as being limited to the representative embodiments described herein. Exemplary embodiments are provided so that this disclosure will be thorough and complete and will fully convey the scope of the present invention to those skilled in the art so that they can make, use, and practice the present invention. In the various drawings, like reference numerals refer to like elements.

[0039] It should be noted that, as used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly indicates otherwise.

[0040] In this specification, concentrations, amounts, and other numerical data may be expressed or presented in a range format. Since such a range format is used only for convenience and brevity purposes, it should be understood that it is to be interpreted flexibly to include not only the numerical values explicitly described as the limits of the range, but also all individual numerical values or sub-ranges included within the range as if they were explicitly described. As an example, a numerical range of "about 1 to 5" should be interpreted to include not only the explicitly described numerical values of "about 1 to about 5", but also the individual numerical values and sub-ranges within that range. Thus, this numerical range includes not only the individual numbers 1, 2, 3, 4, 5, but also individual values such as 2, 3, 4, and sub-ranges such as 1 to 3, 2 to 4, 3 to 5. The same principle applies to ranges that indicate only one numerical value as the minimum or maximum value. Furthermore, such an interpretation should apply regardless of the breadth of the range or the characteristics being described.

[0041] As used herein, "standardization" enables the accurate determination of growth factors and components within the disclosed micronized compositions and sterile micronized compositions for subsequent use and treatment of the subject to be treated (e.g., wound healing treatment). The applicant has determined the range of delivery factors per 1 mg delivered to the patient (subject to be treated) in particulate form. What this means is that the amounts of IL-1ra, HGF, VEGFR1, HA, etc., delivered to the patient / subject to be treated are measured as the amount eluting from the particulates per 1 mg of particulates. The quantification process used for standardizing the disclosed micronized compositions and sterile micronized compositions is used to accurately reflect the relationship between the particulates (mass of the particulates) and the growth factors and other chemical components contained therein. The standardization calculation is determined by the following formula.

Number

[0042] This is achieved by measuring the weight of the sample. The sample is placed in a buffer at 37°C for 48 - 72 hours. The contents are recovered and the desired components are analyzed. The result is multiplied by the amount of buffer recovered. This number is divided by the mg of the sample used. This method can be utilized regardless of the amount of eluate used and the size of the sample. In the tests presented in the present invention, microparticles placed in buffer at 37°C for 72 hours were utilized, mimicking the application of microparticles to internal or external wound spaces. When the eluate was recovered and analyzed, it resembled what had eluted from the microparticles into the wound. As a result, a controlled, relativizable amount of microparticles was tested, and a controlled, relativizable, quantifiable amount of growth factors and stromal components was obtained.

[0043] The "intact human placental tissue" and / or "intact cross-section of placental tissue" as defined herein and further shown in FIGS. 2 - 4 includes the human amniotic layer, the human chorionic layer, and the intact human spongy intermediate layer located between and connecting the human amniotic layer and the human chorionic layer, and immediately prior to micronization, the human amniotic layer and the human chorionic layer are not separated from each other and / or the human spongy intermediate layer is not removed.

[0044] "Cryo-fracture" is a technique that requires the presence of water within the specific cells and / or tissue on which this process is performed. During this process, the cells and / or tissue are rapidly and abruptly cooled (i.e., frozen), and then fractured under vacuum. The water present within the cells and / or tissue forms ice crystals upon rapid cooling, disrupting the cell membranes within the cells and / or tissue on which cryo-fracture is being performed. Taylor, J. (2008), Cryo-Fracture or Freeze-Fracture, a Method to Expose Internal Tissue Surfaces and Cell Surfaces for Viewing in the Scanning Electron Microscope, Microscopy Today, 16(4), 56 - 59, doi:10.1017 / S1551929500059812

[0045] The compositions and methods described herein may comprise, consist of, or consist essentially of the essential elements and limitations described herein, as well as any additional or optional ingredients, components, or limitations described herein. Micronized composition prepared from intact human placental tissue

[0046] Disclosed herein are micronized compositions (and / or sterile micronized compositions) prepared from intact human placental tissue for use in wound healing (e.g., topical wounds, including epidermal and dermal wounds / lesions, oral wounds / lesions, lung and / or tracheal wounds / lesions, etc.). These compositions are minimally processed compared to human placental tissue in vivo, and more advantageously, maintain various endogenous growth factors better compared to conventional compositions / formulations. Further, the growth factor profile of the disclosed compositions is similar to that of the human placenta in vivo (particularly intact human amnion, human chorion, and the intermediate spongy layer located therebetween), thus showing better wound healing properties. The micronized compositions and sterile micronized compositions disclosed herein are (1) usable for irregular wounds and areas and / or wounds where fluid has accumulated (e.g., exudative wounds), (2) by standardizing the growth factors in the composition, the concentration of growth factors administered to the subject by the micronized composition and / or sterile micronized composition during use can be accurately determined, (3) the intermediate spongy layer between the human amnion layer and the human chorion layer (and / or the endogenous growth factors found within the intermediate spongy layer) is preserved, and (4) can provide a number of advantages over currently known compositions in the art, such as being able to quantitatively and / or controllably release IL-1ra, HGF, VEGFR1, HA, glycosaminoglycan, and collagen from the micronized composition and / or sterile micronized composition to the recipient's tissue, but are not limited to these advantages.

[0047] In certain embodiments, the compositions disclosed herein comprise an intact placental tissue (e.g., having an intact cross-section as shown in FIGS. 2-4) comprising a human amniotic layer, a human chorionic layer, and an intact human intermediate sponge layer located between and connecting the human amniotic layer and the human chorionic layer, a micronized composition and / or a sterile micronized composition prepared from the intact placental tissue, wherein, immediately prior to micronization, the human amniotic layer and the human chorionic layer are not separated from each other, so that various endogenous growth factors are maintained therein as compared to the human placenta in vivo. Further, the compositions disclosed herein are advantageously not subjected to harsh processing conditions such as cryogenic disruption and / or lyophilization, and during the preparation of the disclosed compositions, prior to micronization, the human amnion is not physically separated from the human chorion, so that the intermediate sponge layer and the growth factors contained therein are maintained.

[0048] In certain embodiments, disclosed herein is a micronized composition or a sterile micronized composition that is configured for wound healing and prepared from intact human placental tissue, the micronized composition or sterile micronized composition comprising: (a) micronized human amnion; (b) micronized human chorion; and (c) micronized human intermediate spongiosa layer. Among the micronized composition or sterile micronized composition, the micronized human amnion constitutes 10% to 20%, more preferably 10% to 18% of the total weight of the micronized composition or sterile micronized composition. The amnion contains some growth factors at a higher concentration than the chorion. These growth factors include SDF-1, TGF-b1, and GAL-7 (McQuilling JP, Vines JB, Kimmerling KA, Mowry KC, Proteomic Comparison of Amnion and Chorion and Evaluation of the Effects of Processing on Placental Membranes, Wounds: a compendium of clinical research and practice, 2017, 29(6), E36-e40, Epub 2017 / 07 / 07, PubMed PMID: 28682294, PMCID: PMC8009308). Among the micronized composition or sterile micronized composition, the micronized human chorion constitutes 65% to 75%, more preferably 67% to 73% of the total weight of the micronized composition or sterile micronized composition. Since the chorion is relatively thick, it contains most growth factors at a higher concentration than the amnion.For example, the chorion contains a relatively large amount of bFGF, HGF, and PDGF-BB (McQuilling JP, Vines JB, Kimmerling KA, Mowry KC, Proteomic Comparison of Amnion and Chorion and Evaluation of the Effects of Processing on Placental Membranes, Wounds: a compendium of clinical research and practice, 2017, 29(6), E36-e40, Epub 2017 / 07 / 07, PubMed PMID: 28682294, PMCID: PMC8009308). In certain embodiments, the micronized human intermediate sponge layer constitutes 10% to 20%, more preferably 12% to 18%, of the total weight of the micronized composition or the sterile micronized composition. In certain embodiments, the micronized human intermediate sponge layer is particularly rich in type I, type III, type IV collagen, and proteoglycan, compared to micronized human amnion and micronized human chorion, all of which are beneficial components in the wound healing process. Collagen is the most abundant protein in the human body and is a major component of the extracellular matrix. In wound healing, collagen not only attracts fibroblasts and promotes the deposition of new collagen, but also binds to and inactivates excessive matrix metalloproteinases (degrading enzymes). Proteoglycan is a core protein with covalently linked glycosaminoglycan chains. Proteoglycan is a major component of the ECM where proteoglycan forms a complex with HA, collagen, and other matrix proteins. Proteoglycan not only binds to water, potassium, sodium, and calcium, but also affects the movement, stability, and signal transduction of substances within the ECM. In certain embodiments, the micronized human amnion and the micronized human intermediate sponge layer may be present in the micronized composition (and / or the sterile micronized composition) such that the ratio of micronized human amnion:micronized human intermediate sponge layer is 2:1 to 1:2, 1.7:1 to 1:1.5, or 1:1.In certain embodiments, micronized human amnion and micronized human chorion may be present in the micronized composition (and / or sterile micronized composition) such that the ratio of micronized human amnion:micronized human chorion is from 1:7.5 to 1:3.25 and / or may be present in any ratio within these ranges. In certain embodiments, micronized human intermediate sponge layer and micronized human chorion may be present in the micronized composition (and / or sterile micronized composition) such that the ratio of micronized human amnion:micronized human chorion is from 1:7.5 to 1:3.25 and / or may be present in any ratio within these ranges.

[0049] The disclosed micronized composition (and / or sterile micronized composition) prepared from non-damaged human placental tissue contains, in particular, an interleukin-1 receptor antagonist (IL-1ra), a hepatocyte growth factor (HGF), a vascular endothelial growth factor receptor 1 (VEGFR1), hyaluronic acid (HA), glycosaminoglycan (GAG), and collagen, in an amount effective to promote wound healing and the wound healing response of the subject to be treated. Figure 5 is a general schematic diagram showing the endogenous mechanism of action of IL-1ra, HGF, VEGR1, HA, and GAG in vivo and the various signaling pathways in which these growth factors are involved in humans. In particular, IL-1ra non-productively binds to the IL-1 (inflammatory cytokine) receptor, preventing IL-1 (inflammatory molecule) from sending signals. As a result, various IL-1s related to the immune response and inflammatory response are regulated. HGF is a factor for cell proliferation, motility, and morphogenesis secreted by mesenchymal cells. HGF has been found to play a major role in embryonic organogenesis, particularly in myogenesis, adult organ regeneration, and wound healing. Importantly, increased expression of HGF is associated with the enhanced scarless wound healing ability of fibroblasts isolated from oral mucosal tissue. VEGFR1 functions as a cell surface receptor for VEGF-A, VEGF-B, and PGF with respect to embryonic vascular system development, regulation of angiogenesis, cell survival, and cell migration. VEGFR1 can promote the proliferation, survival, and angiogenesis of endothelial cells in adulthood. The function of VEGFR1 to promote cell proliferation seems to be cell type-specific. For example, VEGFR1 promotes the proliferation of endothelial cells via PGF (in vivo), but does not promote the proliferation of normal fibroblasts. Glycosaminoglycan (GAG) is present in all mammalian tissues and interacts with other ECM components in the tissue to organize and form a structural framework suitable for remodeling. GAG also regulates cell growth and proliferation, cell adhesion, anticoagulation, and wound repair. It is well established that hyaluronic acid (HA) is a major component of the extracellular matrix. HA is a non-sulfated glycosaminoglycan and, in particular, provides the backbone of sulfated glycosaminoglycans. HA also binds to integrins to stabilize the ECM and absorbs water, another major component of the ECM.The high molecular weight of HA results in unique biophysical properties such as high viscoelasticity and high colloid osmotic pressure. Furthermore, HA leads to the stabilization of the extracellular matrix, water retention, and control of protein distribution. Collagen is the most abundant protein in the body and is a major component of the extracellular matrix. In wound healing, collagen not only attracts fibroblasts and promotes the deposition of new collagen, but also binds to and inactivates excessive matrix metalloproteinases (degrading enzymes).

[0050] In certain embodiments, the micronized composition or sterile micronized composition comprises at least one of interleukin-1 receptor antagonist (IL-1ra), hepatocyte growth factor (HGF), vascular endothelial growth factor receptor 1 (VEGFR1), hyaluronic acid (HA), glycosaminoglycan (GAG), and collagen in an amount effective to promote wound healing and wound healing response in a subject in need thereof. In certain embodiments, the micronized composition or sterile micronized composition comprises at least two of IL-1ra, HGF, VEGFR1, HA, GAG, and collagen in an amount effective to promote wound healing and wound healing response in a subject in need thereof. In certain embodiments, the micronized composition or sterile micronized composition comprises at least three of IL-1ra, HGF, VEGFR1, HA, GAG, and collagen in an amount effective to promote wound healing and wound healing response in a subject in need thereof. In certain embodiments, the micronized composition or sterile micronized composition comprises at least four of IL-1ra, HGF, VEGFR1, HA, GAG, and collagen in an amount effective to promote wound healing and wound healing response in a subject in need thereof. In certain embodiments, the micronized composition or sterile micronized composition comprises at least five of IL-1ra, HGF, VEGFR1, HA, GAG, and collagen in an amount effective to promote wound healing and wound healing response in a subject in need thereof. In certain embodiments, the micronized composition or sterile micronized composition comprises each of IL-1ra, HGF, VEGFR1, HA, GAG, and collagen in an amount effective to promote wound healing and wound healing response in a subject in need thereof.

[0051] In certain embodiments, the micronized composition or the sterile micronized composition comprises interleukin-1 receptor antagonist (IL-1ra) at 1.0×10 2 to 5.0×10 4 pg / cm 2 , more preferably 1.0×10 2 to 6.0×10 3 pg / cm 2 , hepatocyte growth factor (HGF) at 5.0×10 2 to 1.0×10 4 pg / cm 2 , more preferably 6.0×10 2 to 7.5×10 3 pg / cm 2 , vascular endothelial growth factor receptor 1 (VEGFR1) at 5.0×10 2 to 5.0×10 3 pg / cm 2 , more preferably 8.0×10 2 to 3.0×10 3 pg / cm 2 , hyaluronic acid (HA) at 1.0×10 7 to 1.0×10 8 pg / cm 2 , more preferably 1.5×10 7 to 9.5×10 7 pg / cm 2 , and at least one of glycosaminoglycan (GAG) and collagen, and promotes wound healing and wound healing response in a subject in need thereof. In certain embodiments, the micronized composition or the sterile micronized composition comprises IL-1ra at 1.0×10 2 to 5.0×10 4 pg / cm 2 , more preferably 1.0×10 2 to 6.0×10 3 pg / cm 2 , HGF at 5.0×10 2 to 1.0×10 4 pg / cm 2 , more preferably 6.0×10 2 to 7.5×10 3 pg / cm 2 , VEGFR1 at 5.0×10 2 to 5.0×103 pg / cm 2 and more preferably 8.0×10 2 ~3.0×10 3 pg / cm 2 of VEGFR1, 1.0×10 7 ~1.0×10 8 pg / cm 2 and more preferably 1.5×10 7 ~9.5×10 7 pg / cm 2 of HA, and at least two of glycosaminoglycans (GAGs) and collagen, and promotes wound healing and wound healing response in a subject to be treated. In certain embodiments, the micronized composition or sterile micronized composition is 1.0×10 2 ~5.0×10 4 pg / cm 2 and more preferably 1.0×10 2 ~6.0×10 3 pg / cm 2 of IL-1ra, 5.0×10 2 ~1.0×10 4 pg / cm 2 and more preferably 6.0×10 2 ~7.5×10 3 pg / cm 2 of HGF, 5.0×10 2 ~5.0×10 3 pg / cm 2 and more preferably 8.0×10 2 ~3.0×10 3 pg / cm 2 of VEGFR1, 1.0×10 7 ~1.0×10 8 pg / cm 2 and more preferably 1.5×10 7 ~9.5×10 7 pg / cm 2 of HA, and at least three of glycosaminoglycan (GAG) and collagen, and promotes wound healing and wound healing response in a subject to be treated. In certain embodiments, the micronized composition or sterile micronized composition is 1.0×10 2 ~5.0×10 4 pg / cm 2, more preferably 1.0×10 2 ~6.0×10 3 pg / cm 2 of IL-1ra, and 5.0×10 2 ~1.0×10 4 pg / cm 2 , more preferably 6.0×10 2 ~7.5×10 3 pg / cm 2 of HGF, and 5.0×10 2 ~5.0×10 3 pg / cm 2 , more preferably 8.0×10 2 ~3.0×10 3 pg / cm 2 of VEGFR1, and 1.0×10 7 ~1.0×10 8 pg / cm 2 , more preferably 1.5×10 7 ~9.5×10 7 pg / cm 2 of HA, and contains at least four of glycosaminoglycan (GAG), and collagen, and promotes wound healing and wound healing response of the subject to be treated. In certain embodiments, the micronized composition or the sterile micronized composition is 1.0×10 2 ~5.0×10 4 pg / cm 2 , more preferably 1.0×10 2 ~6.0×10 3 pg / cm 2 of IL-1ra, and 5.0×10 2 ~1.0×10 4 pg / cm 2 , more preferably 6.0×10 2 ~7.5×10 3 pg / cm 2 of HGF, and 5.0×10 2 ~5.0×10 3 pg / cm 2 , more preferably 8.0×10 2 ~3.0×10 3 pg / cm 2 of VEGFR1, and 1.0×10 7 ~1.0×10 8 pg / cm 2, more preferably 1.5×10 7 ~9.5×10 7 pg / cm 2 of HA, and at least five of glycosaminoglycan (GAG) and collagen, and promotes wound healing and wound healing response of the subject to be treated. In certain embodiments, the micronized composition or the sterile micronized composition is 1.0×10 2 ~5.0×10 4 pg / cm 2 , more preferably 1.0×10 2 ~6.0×10 3 pg / cm 2 of IL-1ra, 5.0×10 2 ~1.0×10 4 pg / cm 2 , more preferably 6.0×10 2 ~7.5×10 3 pg / cm 2 of HGF, 5.0×10 2 ~5.0×10 3 pg / cm 2 , more preferably 8.0×10 2 ~3.0×10 3 pg / cm 2 of VEGFR1, 1.0×10 7 ~1.0×10 8 pg / cm 2 , more preferably 1.5×10 7 ~9.5×10 7 pg / cm 2 of HA, and each of glycosaminoglycan (GAG) and collagen, and promotes wound healing and wound healing response of the subject to be treated. Any endpoint of the above ranges functions as a basis for additional small ranges within that range.

[0052] In certain embodiments, the micronized human amnion has a diameter of 1 μm to 500 μm, preferably 10 μm to 300 μm. The micronized human chorion has a diameter of 1 μm to 500 μm, preferably 10 μm to 300 μm. The micronized human intermediate sponge layer has a diameter of 1 μm to 500 μm, preferably 10 μm to 300 μm. Since the human amnion, human chorion, and human intermediate sponge layer are not separated from each other prior to micronization, it should be understood that each of the micronized particles contained within the disclosed compositions may include any combination of micronized human amnion, micronized human chorion, and / or human intermediate sponge layer. For example, the particles disclosed herein may (1) contain only human amnion, (2) contain only human chorion, (3) contain human intermediate sponge layer, (4) contain a combination of human amnion, human chorion, and human intermediate sponge layer, (5) contain a combination of human amnion and human intermediate sponge layer, or (6) contain a combination of human chorion and human intermediate sponge layer as intended.

[0053] To better achieve a consistent therapeutic wound healing effect and a uniform delivery of growth factors to the subject to be treated, the micronized composition or sterile micronized composition is normalized to the weight of the micronized placental tissue. The micronized composition or sterile micronized composition is normalized as follows.

Number

[0054] As described above, the disclosed micronized composition or sterile micronized composition has various wound healing applications, and in particular, is configured for wound packing, treatment of wounds having irregular surfaces and / or surrounding boundaries (e.g., epidermal and / or dermal wounds), exudative wounds, dental and / or oral wounds, or any combination thereof. The particle size, size, and hardness of the disclosed micronized composition and sterile micronized composition enable direct application to the above-mentioned wounds having irregular surfaces and / or surrounding boundaries as well as the above-mentioned exudative wounds. In particular, unlike conventional human amniotic laminates and / or human chorionic laminates (having a flat outer surface), the size and shape of the disclosed micronized composition and sterile micronized composition facilitate attachment to the above-mentioned wounds and subsequent release thereof.

[0055] The micronized composition or sterile micronized composition disclosed herein may further be configured as a therapeutic aerosol configured to be administered intranasally and / or by pulmonary administration to a subject in need thereof. In this aspect, the micronized composition or sterile micronized composition disclosed herein may be contained, for example, within an inhaler and / or a nasal administration device, and may further contain an inert propellant to assist in the pulmonary and / or nasal administration of the micronized composition or sterile micronized composition to the subject in need thereof. The therapeutic aerosol is configured to treat pulmonary wounds, lung lesions, and / or lung disorders, tracheal wounds, tracheal lesions, and / or tracheal disorders, and combinations thereof in the subject in need thereof.

[0056] In certain embodiments, a therapeutic dressing is also disclosed that is configured for topical wound treatment of a subject to be treated (e.g., wounds of the epidermis and dermis) and includes a micronized composition or a sterile micronized composition. The therapeutic dressing includes a cellulose matrix, a polymer matrix, or a combination thereof in which the disclosed micronized composition and / or sterile micronized composition is incorporated. The cellulose matrix includes a carboxyalkylcellulose matrix or a hydroxyalkylcellulose matrix. In certain embodiments, the therapeutic dressing is configured to sustainably release the sterile micronized composition to the wound over a predetermined period. In one aspect, the invention relates to a micronized composition or a sterile micronized composition for use in the treatment of a wound of a human subject to be treated, the treatment including contacting the micronized composition or sterile micronized composition with the wound over a predetermined period to promote healing of the wound of the human subject to be treated. A sterile human placental allograft for use, wherein the micronized composition or sterile micronized composition is transplanted or applied to the wound of the subject to be treated. A micronized composition or a sterile micronized composition for use, wherein the wound is an internal wound and / or a dental wound of the subject to be treated. A micronized composition or a sterile micronized composition for use, wherein the micronized composition or sterile micronized composition is topically applied to the wound of a human subject to be treated. A micronized composition or a sterile micronized composition for use, wherein the wound is a diabetic ulcer. A micronized composition or a sterile micronized composition for use, wherein the wound is a diabetic foot ulcer. Method for producing a micronized composition prepared from intact human placental tissue

[0057] Figure 1 generally discloses a method for generating the micronized compositions (and sterile micronized compositions) disclosed herein from intact human placental tissue (i.e., human amnion, human chorion, and the intermediate spongy layer located between and connecting human amnion and human chorion). The method disclosed herein does not utilize exogenous enzymes in the generation method of the disclosed compositions, and exogenous enzymes are not added to the disclosed micronized compositions (and sterile micronized compositions), thus ensuring that the growth factor profile of the disclosed compositions is similar to the growth factor profiles of human amnion, human chorion, and the intermediate spongy layer therebetween in vivo, and improving the wound healing efficacy of the compositions generated by this method. Further, unlike conventional processes in this field, the lyophilization process and / or the freeze-crushing process are not utilized in the generation of the disclosed compositions. This is because lyophilization (or freeze-drying) and / or freeze-crushing (or freeze-milling) may cause the degradation of various growth factors in the compositions generated by this method, and may ultimately reduce the wound healing power of the compositions generated by this method.

[0058] As specifically shown in FIG. 1, in step (a), within 24 to 96 hours, more preferably within 24 to 72 hours after excision from the donor, fresh intact placental tissue is prepared from a human donor. This fresh intact placental tissue includes intact human amnion, intact human chorion, and an intact intermediate spongy layer located between and connecting the intact human amnion and the intact human chorion. Before and during the implementation of step (a), the fresh intact placental tissue is maintained at a temperature of 4°C to 8°C in order to reduce and / or minimize the degradation of the placental tissue (e.g., by apoptosis, necroptosis, and / or lysosomal degradation). Further, throughout the method disclosed immediately below and further shown in FIG. 1, the temperature is preferably maintained at 4°C to 20°C, more preferably 4°C to 8°C, in order to reduce and / or minimize the degradation of the placental tissue and the compositions obtained from the method.

[0059] After step (a), in step (b), the intact human amnion / chorion having an intact intermediate sponge layer located between and connecting the intact human amnion and the intact chorion in step (a) is rinsed with an alcohol solution for a predetermined time so that excess non-amniotic human placental components and / or other contaminants are washed away. This predetermined time is from 60 seconds to 120 seconds, and in a preferred embodiment, it is 90 seconds. In a particular embodiment, the alcohol solution is preferably isopropyl alcohol at a concentration of 70% to 100%. In a preferred embodiment, the alcohol concentration is 70% to 75%, and in the most preferred embodiment, the alcohol is 70% isopropyl alcohol. Isopropyl alcohol is preferred over other commercially available laboratory and / or pharmaceutical grade alcohols such as ethanol because it can advantageously disinfect and clean the human amnion without damaging the placental tissue (e.g., causing excessive dehydration, inducing an apoptotic process, and / or inducing a necrotic process).

[0060] After step (b), in step (c), the intact human amnion / chorion having an intact intermediate sponge layer located between and connecting the intact human amnion and the intact chorion of step (b) is washed with an isotonic solution to wash away excess non-amniotic human placental components and / or other contaminants. The isotonic solution includes, for example, phosphate buffered saline (PBS) (1xPBS) (or one of lactated Ringer's (NaCl 6g / L, sodium lactate 3.1g / L, KCl 0.3g / L, CaCl 0.2g / L, pH6.5), isotonic saline (0.9wt% NaCl), Plasma-Lyte® (NaCl 5.26g / L, KCl 0.37g / L, magnesium chloride hexahydrate 0.30g / L, sodium acetate trihydrate 3.68g / L, sodium gluconate 5.02g / L, pH7.4), Normosol® (NaCl 5.26g / L, KCl 0.37g / L, magnesium chloride 0.30g / L, sodium acetate anhydrous 2.22g / L, sodium gluconate 5.02g / L, pH7.4)). The washing step of step (c) may be performed only once and / or may be repeated one, two, three, or four times, using a predetermined amount (e.g., 300 mL to 1000 mL, preferably 500 mL) of the isotonic solution at a temperature of 4°C to 20°C, more preferably 4 to 15°C, for 5 to 15 minutes.

[0061] In certain embodiments, prior to step (b), during the performance of step (b), prior to step (c), and / or during the performance of step (c), it may be confirmed whether there are blood clots and / or blood pools in the placental tissue (i.e., amnion, chorion, and spongy intermediate layer), and if so, the blood clots / pools are removed from the intact human amnion / chorion having an intact intermediate spongy layer located between and connecting the intact human amnion and the intact human chorion. If a blood clot is present, the blood clot is removed using suction or other mechanical removal means (e.g., scalpel, forceps, and gauze), such that the immunogenic components (e.g., hemoglobin and / or heme-related components from the human amnion donor) in the finally obtained micronized composition and / or sterile micronized composition are further reduced as much as possible. This avoids and / or minimizes the immunogenic response of the treating agent when the disclosed composition is administered to the treating agent.

[0062] During the performance of steps (b) and (c) and throughout the overall method shown in FIG. 1 (i.e., all steps), it is essential to maintain a sterilized working environment and / or a sterile working environment in order to prevent and / or reduce the contamination by any contaminants during the production of the micronized composition (and / or sterile micronized composition).

[0063] After step (c), in the drying step (d), the intact human amnion / chorion having an intact intermediate sponge layer located between and connecting the intact human amnion and the intact human chorion in step (c) is dried, thereby resulting in a dried intact human amnion / chorion having an intact intermediate sponge layer located between and connecting the intact human amnion and the intact human chorion. In particular, in step (d), the amnion / chorion is laid flat and placed in a biosafety cabinet with a circulation fan at ambient temperature for 2 to 6 hours, or in a dehydrator for 1 to 24 hours, more preferably 4 to 24 hours (at a temperature of 30°C to 40°C). The finally obtained dried intact human amnion / chorion having an intact intermediate sponge layer located between and connecting the intact human amnion and the intact human chorion has a maximum water content of 10% to 15% by weight. This means that the dried intact human amnion / chorion having an intact intermediate sponge layer located between and connecting the intact human amnion and the intact human chorion is in a state insufficient for performing the freeze-crushing process known in the art, and the possibility of the endogenous decomposition process due to the presence of water is further reduced, and the decomposition of growth factors can be further minimized during the implementation of the method of FIG. 1.

[0064] After step (d), the size of the dried intact human amnion / chorion having an intact intermediate sponge layer located between and connecting the intact human amnion and the intact human chorion is further reduced for the subsequent crushing / atomization process. It should be noted that when reducing the size in step (d), the human amnion, the human chorion, and the intermediate sponge layer are not separated from each other. Instead, for example, in the cross-sectional view of the human placental tissue shown in FIGS. 2 to 4, the human chorion and the human amnion remain connected to each other by the intact intermediate sponge layer therebetween and remain intact (especially in the cross-section).

[0065] In step (e), a grinding process is further performed on the small-sized dry intact human amnion / chorion having an intact intermediate sponge layer located between and connecting the intact human amnion and the intact human chorion, resulting in the formation of a micronized composition or a sterile micronized composition configured for wound healing and prepared from intact human placental tissue. In particular, in step (e), a grinding process and / or a cryogenic grinding process (crushing process) configured to obtain particles having sizes of more than 1 μm to 500 μm, more than 1 μm to 400 μm, more than 1 μm to 300 μm, more than 1 μm to 200 μm, more than 1 μm to 100 μm, more than 1 μm to 50 μm, more than 1 μm to 25 μm, more than 50 μm to 500 μm, more than 50 μm to 400 μm, more than 50 μm to 300 μm, more than 50 μm to 200 μm, more than 50 μm to 100 μm, more than 10 μm to 500 μm, more than 10 μm to 400 μm, more than 10 μm to 300 μm, more than 10 μm to 200 μm, more than 10 μm to 100 μm is performed on the small-sized dry intact human amnion / chorion having an intact intermediate sponge layer located between and connecting the intact human amnion and the intact human chorion. Any endpoint within the above ranges also functions as an endpoint of an additional range. Due to the above-described processing conditions, the particles may include various combinations of human amnion, human chorion, and the sponge intermediate layer, and these combinations include: (1) particles having only human amnion, (2) particles having only human chorion, (3) particles having a human intermediate sponge layer, (4) particles having a combination of human amnion, human chorion, and a human intermediate sponge layer, (5) particles having a combination of human amnion and a human intermediate sponge layer, or (6) particles having a combination of human chorion and a human intermediate sponge layer.

[0066] In certain embodiments, the grinding performed in step (e) is, for example, the cryogenic grinding process described in US 20160287749, US 20170203004, and US Patent No. 10,105,398 (each of which is incorporated herein by reference in its entirety), in which a small-sized, dry, intact human amnion / chorion having an intact intermediate spongy layer located between and connecting the intact human amnion and intact human chorion of step (e) is placed in a liquid nitrogen-cooled cryogenic grinding chamber and ground therein to form a micronized composition and a sterile micronized composition prepared from intact human placental tissue that is configured for wound healing and has the above particle size.

[0067] In certain embodiments, the crushing and / or grinding (cryogenic grinding) is continued for 30 seconds to 4 minutes, 30 seconds to 3 minutes, or 1.0 minute to 2.5 minutes per cycle. As further shown in FIG. 1, the method for generating the micronized composition or sterile micronized composition disclosed herein may further comprise step (f), in which the crushing and / or grinding process of step (e) is repeated for a predetermined number of cycles (e.g., an additional 1 cycle, an additional 2 cycles, an additional 3 cycles, an additional 4 cycles, an additional 5 cycles, an additional 6 cycles, etc.), and each of the additional cycles is continued for a predetermined time (e.g., 30 seconds to 4 minutes, 30 seconds to 3 minutes, 1.0 minute to 2.5 minutes). By adding the number of cycles, the particle size of the micronized composition and sterile micronized composition becomes smaller and more uniform.

[0068] Step (g i ) involves placing the micronized composition and sterile micronized composition of step (e) and / or step (f) in a sterile container, sealing it, and storing it at a predetermined temperature for subsequent use on a subject for various therapeutic purposes disclosed herein. Alternatively, step (g iiIn (e) and / or (f), the micronized composition and the sterile micronized composition are placed in a container, sealed, and electron beam sterilized for subsequent use on a subject for various therapeutic purposes disclosed herein.

[0069] As described above, during the preparation of the micronized composition and the sterile micronized composition disclosed herein, the human amnion, human chorion, and intermediate spongiosa layer are not separated from each other. By physically separating the amnion from the chorion, the intermediate spongiosa layer is removed. Different from other compositions in the art that lose a portion of the intermediate spongiosa layer, in the above steps, it is ensured that substantially all of the intermediate spongiosa layer remains in the micronized composition and the sterile micronized composition disclosed herein. The intermediate spongiosa layer contains type I, type III, and type IV collagens and proteoglycans, all of which are components beneficial to the wound healing process. Collagen is the most abundant protein in the human body and is a major component of the extracellular matrix. In wound healing, collagen not only attracts fibroblasts and promotes the deposition of new collagen, but also binds to and inactivates excessive matrix metalloproteinases (degradation). Proteoglycan is a core protein to which glycosaminoglycan chains are covalently bound. Proteoglycan is a major component of the ECM in which proteoglycan forms a complex with HA, collagen, and other matrix proteins. Proteoglycan not only binds to water, sodium, potassium, and calcium, but also affects the movement, stability, and signal transduction of substances within the ECM.

[0070] The foregoing description provides embodiments of the present invention by way of example only. It is also contemplated that other embodiments may perform similar functions and / or achieve similar results. All such equivalent embodiments and examples are within the scope of the present invention and are encompassed by the appended claims. Examples

[0071] The following examples are presented to provide a complete disclosure and description to those skilled in the art of how the compounds, compositions, and methods described herein and claimed in the claims are made and evaluated, and are merely exemplary and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure the accuracy of numerical values (e.g., amounts, temperatures, etc.), but some errors and deviations can occur. Unless otherwise noted, parts are by weight, temperature is in °C or at room temperature if not stated, and pressure is at or near atmospheric pressure. For example, there are numerous variations and combinations of conditions, such as component concentrations, temperatures, pressures, other reaction ranges, and conditions used to optimize the purity and yield of the products obtained from the described processes. Optimizing such process conditions requires only reasonable and routine experimentation. Example 1

[0072] Under informed consent, human placentas were provided after natural or planned cesarean section. All donations and treatments were conducted in accordance with the FDA's Good Tissue Practice (GTP) and the standards of the American Association of Tissue Banks (AATB). Donors were screened for medical problems, social issues, infectious diseases, and infections including human immunodeficiency virus (HIV), human T-lymphotropic virus (HTLV), hepatitis B and C, syphilis, and cytomegalovirus (CMV). The amnion and chorion in the unseparated state, including the intermediate layer, were separated from the placenta and processed. This processing included several gentle washes followed by freezing and dehydration without high heat. The dehydrated amnion / chorion thus obtained was micronized using a vibratory mill, packaged without a carrier in weights of 75 mg, and subjected to electron beam sterilization (Steri-tek, Fremont, California). The dehydrated and micronized amnion / chorion was processed intact without delamination. The finally sterilized product can be administered as a coagulation / healing aid by tapping the powder directly from the vial into an open wound or by removing the powder from the vial using a sterile applicator and transferring it to the wound. Example 2

[0073] The micronized product was made by one steel milling ball in a sterilized 50 mL chamber, which was then sealed. This chamber was placed in a cryogenic mill (CryoMill from Retsch, Haan, Germany), pre-cooled for 5 minutes, and subjected to 4 cycles of 2.5 minutes at 10 cycles per second. The micronized tissue was screened using a 125 μm sieve and a 300 μm sieve that were sterilized according to US ASTM standards. The sterilized sieves were stacked such that the 125 μm sieve was below and the 300 μm sieve was above. The micronized material was transferred from the 50 mL chamber to the 300 μm sieve. This sieve was shaken to separate the micronized particles. If there were particles remaining on the 300 μm sieve, those particles were milled for an additional 30 seconds and sieved again. After sieving the micronized particles, the micronized particles with a particle diameter less than 300 μm were collected, placed in vials by weight (60 mg), and sent for electron beam sterilization (Steri-tek, Fremont, California). Example 3

[0074] All layers were processed without separation, sterilized, micronized, and tested for the growth factor content of the amnion / chorion / intermediate layer that had been dehydrated. The contents of the vial (75 mg) were transferred to one well of a 24-well plate, and 500 μl of calcium- and magnesium-free DPBS was added to that well. To avoid the effect of evaporation, the plate was covered and sealed with parafilm. Then, the plate was placed on a rocking plate in an incubator set at 37 °C for 72 hours. From previous studies (unpublished) by the present inventors, it has been found that elution from the sample is incomplete if it is less than 72 hours, and complete elution is achieved if it exceeds 72 hours. Even if it is longer than 72 hours, it will not be advantageous for the assay and will only promote the degradation of growth factors. After 72 hours, the contents of the well were collected and centrifuged at 10,000 g for 10 minutes to purify the sample. All of the supernatant was used for all assays. For all assays, five separate lots were performed in duplicate. For the determination of IL-1ra, HGF, and VEGFR1, the supernatant was diluted 1:10 with the sample diluent for the BioPlex Pro single plex cytokine assay (Bio-Rad, Hercules, CA). For the determination of HA, the sample was diluted 1:2000 with the sample diluent for the hyaluronic acid immunoassay (R&D Systems, Minneapolis, MN). The amount of the eluate (500 μl) was multiplied by the results of the assay (unit: pg / ml) to obtain the total pg of each factor in the sample. The total pg was divided by the mg (75 mg) of the sample used in the assay to obtain the amount of each factor (pg / mg) in each mg of the sample. The standard error of the mean (SEM) for reporting was obtained by dividing the standard deviation by the square root of the number of samples (5 lots). This information provided for the microparticles may be considered if the user wishes to use 30 mg or 100 mg of microparticles. Furthermore, this method and calculation are the best in vivo methods to reflect the availability to patients of any factor measured from the microparticles.

Claims

**Claim 1** A sterile micronized composition configured for wound healing and prepared from intact human placental tissue, comprising: (a) micronized human amnion; (b) micronized human chorion; (c) micronized human intermediate sponge layer. **Claim 2** The sterile micronized composition according to claim 1, wherein the micronized human amnion constitutes 10% to 20% of the total weight of the sterile micronized composition. **Claim 3** The sterile micronized composition according to claim 1 or 2, wherein the micronized human chorion constitutes 65% to 75% of the total weight of the sterile micronized composition. **Claim 4** The sterile micronized composition according to any one of claims 1 to 3, wherein the micronized human intermediate sponge layer constitutes 10% to 20% of the total weight of the sterile micronized composition. **Claim 5** The composition further comprises at least two of: (a) 1.0 × 10 ２ ~5.0 × 10 ４ pg / cm ２ of interleukin-1 receptor antagonist (IL-1ra), and (b) 5.0 × 10 ２ ~ 1.0 × 10 ４ pg / cm ２ of hepatocyte growth factor (HGF), and (c) 5.0 × 10 ２ ~ 5.0 × 10 ３ pg / cm ２ of vascular endothelial growth factor receptor 1 (VEGFR1), and (d) 1.0 × 10 ７ ~1.0 × 10 ８ pg / cm ２ of hyaluronic acid (HA), and (e) glycosaminoglycan (GAG); (f) collagen. The sterile micronized composition according to any one of claims 1 to 4. **Claim 6** The composition further comprises at least three of: (a) 1.0 × 10 ２ ~5.0 × 10 ４ pg / cm ２ of IL-1ra, and (b) 5.0 × 10 ２ ~1.0 × 10 ４ pg / cm ２ of HGF, and (c) 5.0 × 10 ２ ~ 5.0 × 10 ３ pg / cm ２ of VEGFR1, and (d) 1.0 × 10 ７ to 1.0 × 10 ８ pg / cm ２ of HA, and (e) GAG; (f) collagen. The sterile micronized composition according to any one of claims 1 to 5. **Claim 7** The composition further comprises at least four of: (a) 1.0×10 ２ ~5.0×10 ４ pg / cm ２ of IL-1ra, and (b) 5.0 × 10 ２ ~1.0 × 10 ４ pg / cm ２ of HGF, and (c) 5.0 × 10 ２ to 5.0 × 10 ３ pg / cm ２ of VEGFR1, and (d) 1.0×10 ７ ~1.0×10 ８ pg / cm ２ of HA, and (e) GAG; (f) collagen. The sterile micronized composition according to any one of claims 1 to 6. **Claim 8** The composition further comprises at least five of: (a) 1.0×10 ２ ~ 5.0×10 ４ pg / cm ２ of IL-1ra, and (b) 5.0 × 10 ２ ~ 1.0 × 10 ４ pg / cm ２ of HGF, and (c) 5.0 × 10 ２ ~5.0 × 10 ３ pg / cm ２ of VEGFR1, and (d) 1.0×10 ７ ~1.0×10 ８ pg / cm ２ of HA, and (e) GAG; (f) collagen. The sterile micronized composition according to any one of claims 1 to 7. **Claim 9** The composition comprises each of: (a) 1.0×10 ２ ~5.0×10 ４ pg / cm ２ of IL-1ra, and (b) 5.0 × 10 ２ ~ 1.0 × 10 ４ pg / cm ２ of HGF, and (c) 5.0 × 10 ２ to 5.0 × 10 ３ pg / cm ２ of VEGFR1, and (d) 1.0×10 ７ ~1.0×10 ８ pg / cm ２ of HA, and (e) GAG; (f) collagen. The sterile micronized composition according to any one of claims 1 to 8. **Claim 10** The sterile micronized composition according to any one of claims 1 to 9, wherein the micronized human amnion has a diameter of 1 μm to 500 μm. **Claim 11** The sterile micronized composition according to any one of claims 1 to 10, wherein the micronized human chorion has a diameter of 1 μm to 500 μm. **Claim 12** The sterile micronized composition according to any one of claims 1 to 11, wherein the micronized human intermediate sponge layer has a diameter of 1 μm to 500 μm. **Claim 13** The sterile micronized composition according to any one of claims 1 to 12, wherein the micronized composition is not cryogenically crushed. **Claim 14** The composition comprises: a human amnion layer; a human chorion layer; an intact human intermediate sponge layer located between the human amnion layer and the human chorion layer and connecting the human amnion layer and the human chorion layer. ​ It is prepared from placental tissue with an intact cross-section, The sterile micronized composition according to any one of claims 1 to 13, wherein the human amniotic layer and the human chorionic layer are not separated from each other immediately before micronization.

15. The sterile micronized composition according to claim 14, wherein the sterile micronized composition is sterilized, dehydrated, and standardized to the weight of the micronized placental tissue.

16. The sterile micronized composition according to claim 15, wherein the sterile micronized composition is standardized as follows. 【Number 1】

17. The sterile micronized composition according to any one of claims 1 to 16, wherein the micronized composition is configured for wound packing, treatment of wounds having irregular surfaces and / or surrounding boundaries, exudative wounds, dental wounds and / or oral wounds and / or incisions, or any combination thereof.

18. A pharmaceutical product comprising the composition according to claim 1.

19. A therapeutic aerosol comprising the composition according to claim 1, configured to be administered intranasally and / or by pulmonary administration to a subject in need of treatment.

20. The therapeutic aerosol according to claim 19, configured to treat lung disorders, tracheal disorders, and combinations thereof.

21. A therapeutic bandage configured for local wound treatment of a subject in need of treatment, comprising the composition according to claim 1.

22. The therapeutic bandage according to claim 21, wherein the therapeutic bandage comprises a cellulose matrix, a polymer matrix, or a combination thereof in which the sterile micronized composition is incorporated.

23. The therapeutic bandage according to claim 22, wherein the cellulose matrix comprises a carboxyalkyl cellulose matrix or a hydroxyalkyl cellulose matrix.

24. The therapeutic bandage according to any one of claims 21 to 23, wherein the therapeutic bandage is configured to sustainably release the sterile micronized composition to a wound over a predetermined period of time.

25. The sterile micronized composition according to any one of claims 1 to 17, wherein the sterile micronized composition is sterilized by electron beam sterilization.

Images