Multi-part processed human amniotic membrane composition for treating Peyronie's disease and methods of making and using same - Patent Application 20070123333

Abstract

A multi-part processed human amniotic membrane composition configured to treat Peyronie's disease in a subject by injecting the composition internally into the corpus cavernosum of the subject in need of treatment to reduce plaque size associated with Peyronie's disease. The multi-part processed human amniotic membrane composition includes a micronized human amniotic membrane composition and an aqueous human amniotic fluid filtrate configured to reconstitute and suspend the micronized human amniotic membrane composition. In certain embodiments, the multi-part processed human amniotic membrane composition is not treated with exogenous enzymes during its manufacture and does not include added exogenous enzymes such as collagenase.

Description

[Technical field]

[0001] The present invention relates generally to the field of compositions derived from amniotic membrane, and more specifically to a non-immunogenic, multi-part processed human amniotic membrane composition for treating Peyronie's Disease, which is produced directly from human amniotic membrane and amniotic fluid without the use of exogenous enzymes and / or does not contain added collagenase or the like / exogenous enzymes. [Background technology]

[0002] The human penis is the external segment of the male reproductive system consisting of three chambers of spongy tissue surrounded by the tunica albuginea.

[0003] During sexual arousal, a physiological response occurs in which the penile tissue fills with blood perfused from elsewhere in the body, causing increased penile rigidity. Several penile disorders, including erectile dysfunction (ED) and Peyronie's disease (PD), affect and / or prevent this physiological response. ED is generally defined as the inability to obtain and / or maintain an erection for sexual intercourse. ED is common, affecting approximately half of American men over the age of 40. Causes of ED include vascular disorders, neurological disorders, psychological stress, penile injury, chronic disease, and unhealthy lifestyle choices.

[0004] In addition to the causes of ED mentioned above, ED can also be due to Peyronie's disease, which is the formation of plaques (i.e., scar tissue) inside the tunica albuginea of ​​the penis. When the plaques are numerous and / or cover a large surface area within the penis, they can cause painful, curved erections, which often result in ED that makes sexual intercourse very painful, difficult, and / or impossible.

[0005] In the United States, approximately 1 in 100 men over the age of 18 are diagnosed with Peyronie's disease, and the likelihood of developing Peyronie's disease increases with age. A significant number of men with Peyronie's disease develop erectile dysfunction. The exact cause of PD is unknown, but it is theorized to result from injury.

[0006] Scarring (plaque) may develop in the elastic layer surrounding the erectile tissue, which reduces the elasticity of the penis in the affected areas. Symptoms of PD include scar tissue felt under the skin of the penis, significant bending or curvature of the penis, difficulty in achieving or maintaining an erection, penile pain, and shortening of the penis. Surgical treatments include removal of the plaque, shortening of the tissue opposite the plaque to even out the curvature, and penile implants. These procedures carry the risk of lack of erectile function or permanent shortening of the penis.

[0007] Non-surgical treatments use injections of various compositions to soften the plaque and straighten the curvature. These compositions currently include XIAFLEX®, which is made from collagenase derived from bacteria with the goal of dissolving the collagen in the plaque. XIAFLEX® carries the risk of corporal lacerations, penile hematoma, erectile dysfunction, and hematuria. XIAFLEX®. Verapamil is a calcium channel inhibitor used to stop the progression of plaque formation. Side effects include dizziness, weakness, nausea, and sweating. Verapamil also has a long list of drug interactions. Steroid injections have been used for many years. Many patients find the injections painful. Side effects of corticosteroids include a weakened immune system, infection at the injection site, reduced size of penile tissue, thinning of the skin, and complications of future surgical procedures. Radiation therapy is only effective if administered early in PD and is used to stop the progression of PD. Studies of this treatment lack well-controlled and it exposes reproductive tissues to radiation, making many patients hesitant. Summary of the Invention

[0008] In view of the above problems associated with Peyronie's disease treatment, there exists a need for a treatment for penile defects (e.g., Peyronie's disease) that can eliminate penile defects while minimizing side effects. The compositions and methods disclosed herein achieve this goal by utilizing the multi-part processed human amniotic membrane composition disclosed herein as an injectable internal treatment for Peyronie's disease for subjects in need of treatment, maximizing the elimination of penile defects while minimizing side effects and injection discomfort. Additionally, the disclosed multi-part processed human amniotic membrane composition is not treated with exogenous enzymes during its manufacture and does not contain added exogenous enzymes such as collagenase, and therefore preferably maintains an endogenous profile similar to that found in human amniotic fluid and human amniotic tissue (e.g., in vivo), particularly compared to amniotic membrane isolates and / or other PD treatment agents. The disclosed compositions advantageously minimize and / or completely eliminate immunogenic responses and / or other side effects seen with conventional Peyronie's disease treatments by mimicking, containing, and / or retaining a cellular and / or extracellular profile resembling the endogenous profile of human amniotic membrane and human amniotic fluid (e.g., in vivo).

[0009] Disclosed herein is a multi-part processed human amniotic membrane composition configured to treat Peyronie's disease by internal injection of an effective amount into a subject in need of treatment. The multi-part processed human amniotic membrane composition includes (a) a micronized human amniotic membrane composition and (b) an aqueous human amniotic fluid filtrate configured to resuspend the micronized human amniotic membrane composition therein, wherein the multi-part processed human amniotic membrane composition was not treated with exogenous enzymes during its manufacture and does not include added exogenous enzymes (e.g., collagenase).

[0010] In certain embodiments, the micronized human amniotic membrane composition is dried or comminuted micronized human amniotic membrane tissue.

[0011] In certain embodiments, the micronized human amniotic membrane composition has a particle diameter size of greater than 1 μm to less than 300 μm.

[0012] In certain embodiments, the micronized human amniotic membrane composition has a particle diameter size of greater than 1 μm to 50 μm and the particles are polydisperse. In alternative embodiments, the micronized human amniotic membrane composition has a particle diameter size of greater than 1 μm to 50 μm and the particles are substantially monodisperse.

[0013] In certain embodiments, the micronized human amniotic membrane composition has a particle diameter size of greater than 1 μm to 25 μm and the particles are polydisperse. In alternative embodiments, the micronized human amniotic membrane composition has a particle diameter size of greater than 1 μm to 25 μm and the particles are monodisperse.

[0014] In certain embodiments, the micronized human amniotic membrane composition comprises hyaluronic acid (HA) and / or hyaluronan, fibronectin, insulin growth factor binding protein-1 (IGFBP-1), sulfated glycosaminoglycans (sGAGs), exosomes, interleukin-1 receptor antagonist (IL-1ra), hepatocyte growth factor (HGF), transthyretin, or any combination thereof.

[0015] In certain embodiments, the micronized human amniotic membrane composition is dried micronized human amniotic membrane tissue having a particle diameter size of greater than 1 μm to less than 300 μm and comprises hyaluronic acid (HA) and / or hyaluronan, fibronectin, insulin growth factor binding protein-1 (IGFBP-1), sulfated glycosaminoglycans (sGAGs), exosomes, interleukin-1 receptor antagonist (IL-1ra), hepatocyte growth factor (HGF), transthyretin, or any combination thereof.

[0016] In certain embodiments, the aqueous human amniotic fluid filtrate comprises hyaluronic acid (HA) and / or hyaluronan, fibronectin, insulin growth factor binding protein-1 (IGFBP-1), sulfated glycosaminoglycans (sGAGs), exosomes, interleukin-1 receptor antagonist (IL-1ra), hepatocyte growth factor (HGF), transthyretin, or combinations thereof.

[0017] In certain embodiments, the aqueous human amniotic fluid filtrate further comprises an isotonic fluid, hi certain embodiments, the isotonic fluid is phosphate buffered saline.

[0018] In certain embodiments, the aqueous human amniotic fluid filtrate contains particles therein that are less than 70 μm in diameter.

[0019] In certain embodiments, the multi-part processed human amniotic membrane composition comprises 1.2×10 micronized human amniotic membrane composition reconstituted and / or suspended in aqueous human amniotic fluid filtrate. 6 ~1.1×10 8 HA at a concentration of 2.5 x 10 pg / mL 6 ~9×10 6 pg / mL IGFBP-1, 5 × 10 7 ~8.0×10 7 pg / mL of sGAG, particle diameter of 50-200 nm, and 1.0 × 10 9 ~2.0×10 9 Exosomes at a concentration of 2 × 10 4 ~1.5×10 5 pg / mL IL-1ra, 900-3000pg / mL HGF, 1.25×10 4 ~2.25×10 4 pg / mL transthyretin, or any combination thereof.

[0020] In certain embodiments, the multi-part processed human amniotic membrane composition comprises 1.2×10 micronized human amniotic membrane composition reconstituted and / or suspended in aqueous human amniotic fluid filtrate. 6 ~1.1×10 8 HA at a concentration of pg / mL and 2.5 × 10 6 ~9×10 6 pg / mL IGFBP-1 and 5 × 10 7 pg / mL ~ 8.0 × 10 7 pg / mL of sGAG and a particle diameter of 50-200 nm, with a concentration of 1.0 × 10 9 ~2.0×10 9 and 2 × 10 4 ~1.5×10 5pg / mL IL-1ra, 900-3000pg / mL HGF, and 1.25×10 4 ~2.25×10 4 and transthyretin in pg / mL.

[0021] In certain embodiments, the multi-part processed human amniotic membrane composition comprises a 1 cm 2 : 1ml~2cm 2 The micronized human amniotic membrane composition is configured to be mixed in a ratio of 1:1 ml and aqueous human amniotic fluid filtrate. In certain embodiments, the multi-part processed human amniotic membrane composition includes a cryopreservation agent therein, the cryopreservation agent having a concentration of 1-10 wt% of the total concentration of the multi-part processed human amniotic membrane composition. For example, in certain embodiments, the cryopreservation agent may be dimethyl sulfoxide (DMSO) present in the multi-part processed human amniotic membrane composition at a total concentration of 1-10 wt% of the multi-part processed human amniotic membrane composition. Alternatively, the cryopreservation agent may be a DMSO-free cryopreservation agent (e.g., polyampholate, P24, etc.) at a total concentration of 1-10 wt% of the multi-part processed human amniotic membrane composition.

[0022] In certain embodiments, both the micronized human amniotic membrane composition and the aqueous human amniotic fluid filtrate are sterile and / or preserved.

[0023] In certain embodiments, both the micronized human amniotic membrane composition and the aqueous human amniotic fluid filtrate are non-immunogenic.

[0024] In certain embodiments, the multi-part processed human amniotic membrane composition reduces the size of Peyronie's disease plaques.

[0025] In certain embodiments, transthyretin is present in the multi-part processed human amniotic membrane composition in an effective amount for treating Peyronie's disease, reducing symptoms associated with Peyronie's disease, and / or reducing the size of Peyronie's disease plaques.

[0026] In certain embodiments, the multi-part processed human amniotic membrane composition contains transthyretin at a concentration of 1.25×10 4 ~2.25×10 4 It is present at concentrations of pg / mL.

[0027] Also disclosed herein is a kit including a multi-part processed human amniotic membrane composition configured to treat Peyronie's Disease upon internal injection of an effective amount into a subject in need of treatment, the kit including a sterile container storing a multi-part processed human amniotic membrane composition configured to treat Peyronie's Disease upon internal injection, the multi-part processed human amniotic membrane composition configured to treat Peyronie's Disease upon internal injection, the multi-part processed human amniotic membrane composition including a micronized human amniotic membrane composition suspended in an aqueous human amniotic fluid filtrate, but not including added exogenous enzymes.

[0028] In certain embodiments, the multi-part processed human amniotic membrane composition adapted to treat Peyronie's Disease by internal injection does not contain collagenase.

[0029] In certain embodiments, the micronized human amniotic membrane composition is dried or comminuted micronized human amniotic membrane tissue, preferably not subjected to exogenous enzymatic digestion.

[0030] In certain embodiments, the micronized human amniotic membrane composition has a particle diameter size of greater than 1 μm to less than 300 μm.

[0031] In certain embodiments, the micronized human amniotic membrane composition has a particle diameter size of greater than 1 μm to 50 μm, and the particles are polydisperse.

[0032] In certain embodiments, the micronized human amniotic membrane composition has a particle diameter size of greater than 1 μm to 25 μm, and the particles are polydisperse.

[0033] In certain embodiments, the micronized human amniotic membrane composition comprises hyaluronic acid (HA) and / or hyaluronan, fibronectin, insulin growth factor binding protein-1 (IGFBP-1), sulfated glycosaminoglycans (sGAGs), exosomes, interleukin-1 receptor antagonist (IL-1ra), hepatocyte growth factor (HGF), transthyretin, or any combination thereof.

[0034] In certain embodiments, the micronized human amniotic membrane composition comprises hyaluronic acid (HA) and / or hyaluronan, fibronectin, insulin growth factor binding protein-1 (IGFBP-1), sulfated glycosaminoglycans (sGAGs), exosomes, interleukin-1 receptor antagonist (IL-1ra), hepatocyte growth factor (HGF), and transthyretin.

[0035] In certain embodiments, the micronized amniotic membrane composition is dried micronized human amniotic membrane tissue having a particle diameter of greater than 1 μm to less than 300 μm and comprises hyaluronic acid (HA) and / or hyaluronan, fibronectin, insulin growth factor binding protein-1 (IGFBP-1), sulfated glycoaminoglycans (sGAGs), exosomes, interleukin-1 receptor antagonist (IL-1ra), hepatocyte growth factor (HGF), transthyretin, or any combination thereof.

[0036] In certain embodiments, the aqueous human amniotic fluid filtrate comprises hyaluronic acid (HA) and / or hyaluronan, fibronectin, insulin growth factor binding protein-1 (IGFBP-1), sulfated glycosaminoglycans (sGAGs), exosomes, interleukin-1 receptor antagonist (IL-1ra), hepatocyte growth factor (HGF), transthyretin, or combinations thereof.

[0037] In certain embodiments, the aqueous human amniotic fluid filtrate further comprises an isotonic fluid.

[0038] In certain embodiments, the isotonic solution is phosphate buffered saline.

[0039] In certain embodiments, the aqueous human amniotic fluid filtrate contains particles therein that are less than 100 μm in diameter.

[0040] In certain embodiments, the micronized human amniotic membrane composition and the aqueous human amniotic fluid filtrate are each 1 cm 2 : 1ml~2cm 2 :1ml. In certain embodiments, the multi-part processed human amniotic membrane composition includes a cryopreservation agent therein, the cryopreservation agent having a concentration of 1-10 wt% of the total concentration of the multi-part processed human amniotic membrane composition. For example, in certain embodiments, the cryopreservation agent may be dimethyl sulfoxide (DMSO) present in the multi-part processed human amniotic membrane composition at a total concentration of 1-10 wt% of the multi-part processed human amniotic membrane composition. Alternatively, the cryopreservation agent may be a DMSO-free cryopreservation agent (e.g., polyampholate, P24, etc.) at a total concentration of 1-10 wt% of the multi-part processed human amniotic membrane composition.

[0041] In certain embodiments, the multi-part processed human amniotic membrane composition configured to treat Peyronie's disease by internal injection contains 1.2×10 6 ~1.1×10 8 HA at a concentration of 2.5 x 10 pg / mL 6 ~9×10 6 pg / mL IGFBP-1, 5 × 10 7 pg / mL ~ 8.0 × 10 7 pg / mL of sGAG, particle diameter of 50-200 nm, and 1.0 × 10 9 ~2.0×10 9 Exosomes at a concentration of 2 × 10 4 ~1.5×10 5 pg / mL IL-1ra, 900-3000pg / mL HGF, 1.25×10 4 ~2.25×10 4 pg / mL transthyretin, or any combination thereof.

[0042] In certain embodiments, the multi-part processed human amniotic membrane composition configured to treat Peyronie's disease by internal injection contains 1.2×10 6 ~1.1×10 8 HA at a concentration of pg / mL and 2.5 × 10 6 ~9×10 6 pg / mL IGFBP-1 and 5 × 10 7 pg / mL ~ 8.0 × 10 7 pg / mL of sGAG and a particle diameter of 50-200 nm, with a concentration of 1.0 × 10 9 ~2.0×10 9 and 2 × 10 4 ~1.5×10 5 pg / mL IL-1ra, 900-3000pg / mL HGF, and 1.25×10 4 ~2.25×10 4 and transthyretin in pg / mL.

[0043] In certain embodiments, the multi-part processed human amniotic membrane composition configured to treat Peyronie's Disease is preservative and / or sterile.

[0044] In certain embodiments, the multi-part processed human amniotic membrane composition adapted to treat Peyronie's Disease is non-immunogenic.

[0045] In certain embodiments, the multi-part processed human amniotic membrane composition configured to treat Peyronie's Disease is for alleviating symptoms associated with Peyronie's Disease and / or for reducing the size of Peyronie's Disease plaques.

[0046] In certain embodiments, transthyretin is present in the multi-part processed human amniotic membrane composition in an amount effective to treat Peyronie's Disease, to alleviate symptoms associated with Peyronie's Disease, and / or to reduce the size of Peyronie's Disease plaques.

[0047] In certain embodiments, the transthyretin is present in a multi-part processed human amniotic membrane composition at a concentration of 1.25×10 4 ~2.25×10 4 It is present at concentrations of pg / mL.

[0048] Also disclosed herein is a method of treating Peyronie's Disease in a subject by (a) providing a multi-part processed human amniotic membrane composition configured to treat Peyronie's Disease as disclosed herein; and (b) injecting an effective amount of the multi-part processed human amniotic membrane composition configured to treat Peyronie's Disease into the corpus cavernosum of a subject in need of treatment, thereby treating Peyronie's Disease, alleviating symptoms associated with Peyronie's Disease, and / or reducing the size of plaques associated with Peyronie's Disease in the subject in need of treatment.

[0049] In certain embodiments, the method comprises administering / injecting a multi-part processed amniotic membrane composition configured to treat Peyronie's disease to a subject by internal injection, the multi-part processed amniotic membrane composition comprising 1.2×10 6 ~1.1×10 8 HA at a concentration of 2.5 x 10 pg / mL 6 ~9×10 6 pg / mL IGFBP-1, 5 × 10 7 ~8.0×10 7 pg / mL of sGAG, particle diameter of 50-200 nm, and 1.0 × 10 9 ~2.0×10 9 Exosomes at a concentration of 2 × 10 4 ~1.5×10 5 pg / mL IL-1ra, 900-3000pg / mL HGF, 1.25×10 4 ~2.25×10 4 pg / mL transthyretin, or any combination thereof, with a total volume of 250 μL to 5 mL per dose / injection.

[0050] In certain embodiments, the method comprises administering / injecting a multi-part processed amniotic membrane composition configured to treat Peyronie's disease to a subject by internal injection, the multi-part processed amniotic membrane composition comprising 1.2×10 6 ~1.1×10 8 HA at a concentration of pg / mL and 2.5 × 10 6 ~9×10 6 pg / mL IGFBP-1 and 5 × 10 7 ~8.0×10 7 pg / mL of sGAG and a particle diameter of 50-200 nm, with a concentration of 1.0 × 10 9 ~2.0×10 9 and 2 × 10 4 ~1.5×10 5 pg / mL IL-1ra, 900-3000pg / mL HGF, and 1.25×10 4 ~2.25×10 4 It contains pg / mL of transthyretin and the total volume per dose / injection is 250 μL to 5 mL.

[0051] Also disclosed herein is a method of making a multi-part processed human amniotic membrane composition configured to treat Peyronie's disease by internal injection into a subject in need of treatment, the method comprising: (a) providing a fresh human amniotic membrane; (b) separating the human amniotic membrane from other human placental sites; (c) rinsing the separated human amniotic membrane of step (b) with alcohol; (d) washing the separated human amniotic membrane of step (c) with an isotonic solution; (e) drying the separated human amniotic membrane of step (d) in a biosafety cabinet with a circulating fan at ambient temperature for 2-6 hours or in a dehydrator (at a temperature of 30° C.-40° C.) for 15 minutes-24 hours to produce a dried human amniotic membrane; and (f) milling the dried human amniotic membrane to produce a micronized human amniotic membrane composition having a particle diameter of 1-500 microns. (a') preparing fresh human amniotic fluid; (b') filtering the fresh human amniotic fluid through a 200 micron filter to produce a first human amniotic fluid filtrate; (c') filtering the first human amniotic fluid filtrate through a 100 micron filter to produce a second human amniotic fluid filtrate; (d') filtering the second human amniotic fluid filtrate through a 70 micron filter to produce a third human amniotic fluid filtrate; (f') optionally diluting the third human amniotic fluid filtrate with a predetermined amount of a balanced salt solution to produce a diluted third human amniotic fluid filtrate; and (i) mixing a micronized human amniotic membrane composition with either the third human amniotic fluid filtrate or the diluted third human amniotic fluid filtrate to produce a multi-part processed human amniotic membrane composition configured to treat Peyronie's disease by internal injection, wherein none of the steps include the introduction of an exogenous enzyme.

[0052] In certain embodiments, fresh human amniotic membrane is obtained from a subject, and steps (a)-(e) are then performed within 48-72 hours after birth and / or cesarean section.

[0053] In certain embodiments, the method further comprises removing a blood clot present in the human amniotic membrane during steps (a)-(c).

[0054] In certain embodiments, step (d) is repeated 1 to 5 times by discarding the used isotonic solution, preparing a new isotonic solution, and washing the human amniotic membrane again with the new isotonic solution.

[0055] In certain embodiments, the isotonic solution is phosphate buffered saline provided in a volume of between 300 mL and 1000 mL per wash step (d).

[0056] In certain embodiments, in step (f), the dried human amniotic membrane is milled using a milling tool configured to grind and / or mince the dried human amniotic membrane at between 40 and 200 revolutions per minute (RPM) until the dried human amniotic membrane is comminuted to produce a micronized human amniotic membrane composition having a particle diameter of between 1 and 500 microns.

[0057] In certain embodiments, the multi-part processed human amniotic membrane composition configured to treat Peyronie's disease by internal injection contains 1.2×10 6 ~1.1×10 8 HA at a concentration of 2.5 x 10 pg / mL 6 ~9×10 6 pg / mL IGFBP-1, 5 × 10 7 ~8.0×10 7 pg / mL of sGAG, particle diameter of 50-200 nm, and 1.0 × 10 9 ~2.0×10 9 Exosomes at a concentration of 2 × 10 4 ~1.5×10 5 pg / mL IL-1ra, 900-3000pg / mL HGF, 1.25×10 4 ~2.25×10 4 pg / mL transthyretin, or any combination thereof.

[0058] In certain embodiments, the multi-part processed human amniotic membrane composition configured to treat Peyronie's disease by internal injection contains 1.2×10 6 ~1.1×10 8 HA at a concentration of pg / mL and 2.5 × 10 6 ~9×106 pg / mL IGFBP-1 and 5 × 10 7 ~8.0×10 7 pg / mL of sGAG and a particle diameter of 50-200 nm, with a concentration of 1.0 × 10 9 ~2.0×10 9 and 2 × 10 4 ~1.5×10 5 pg / mL IL-1ra, 900-3000pg / mL HGF, and 1.25×10 4 ~2.25×10 4 and transthyretin in pg / mL.

[0059] In certain embodiments, transthyretin is present in the multi-part processed human amniotic membrane composition in an amount effective to treat Peyronie's Disease, to alleviate symptoms associated with Peyronie's Disease, and / or to reduce the size of Peyronie's Disease plaques.

[0060] In certain embodiments, the multi-part processed human amniotic membrane composition contains transthyretin at a concentration of 1.25×10 4 ~2.25×10 4 It is present at concentrations of pg / mL.

[0061] Additional features, aspects, and advantages of the present invention will be set forth in the following detailed description, and in part will be readily apparent to those skilled in the art from the description, or may be learned by the practice of the 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 invention, and are intended to provide an overview or framework for understanding the nature and characteristics of the invention as claimed. The accompanying drawings are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification. [Brief description of the drawings]

[0062] These and other features, aspects and advantages of the present invention will be better understood from the following detailed description of the invention when read in conjunction with the accompanying drawings.

[0063] [Figure 1] FIG. 1 is a schematic diagram of the steps involved in manufacturing a micronized human amniotic membrane composition of a multi-part processed amniotic membrane composition for the treatment of Peyronie's Disease.

[0064] [Diagram 2] FIG. 2 is a schematic diagram of the steps involved in producing the aqueous human amniotic fluid filtrate contained in a multi-part processed human amniotic membrane composition for the treatment of Peyronie's Disease.

[0065] [Diagram 3] FIG. 3 is a schematic diagram of the steps involved in mixing the micronized human amniotic membrane composition of FIG. 1 with the amniotic fluid filtrate of FIG. 2 to produce a multi-part processed human amniotic membrane composition having a desired concentration profile of HA, sGAG, IGFBP-A, IL-1ra, HGF, and transthyretin in a predetermined amount for internal administration to a subject in need of treatment and for treating Peyronie's disease.

[0066] [Figure 4] FIG. 4 is a photograph of a multi-part processed human amniotic membrane composition placed in a sterile container according to step (j) of FIG. 3 for the treatment of Peyronie's disease.

[0067] [Diagram 5] FIG. 5 is a graph showing the concentration of HA in a multi-part processed human amniotic membrane composition according to step (j) of FIG. 3 for the treatment of Peyronie's disease.

[0068] [Figure 6] FIG. 6 is a graph showing the concentration of sGAG in a multi-part processed human amniotic membrane composition according to step (j) of FIG. 3 for the treatment of Peyronie's disease.

[0069] [Figure 7] FIG. 7 is a graph showing the concentration of IL-1RA in a multi-part processed human amniotic membrane composition according to step (j) of FIG. 3 for the treatment of Peyronie's disease.

[0070] [Figure 8] FIG. 8 is a graph showing the concentration of HGF in a multi-part processed human amniotic membrane composition according to step (j) of FIG. 3 for the treatment of Peyronie's disease.

[0071] [Figure 9] FIG. 9 is a graph showing the concentration of IGF-BP1 in a multi-part processed human amniotic membrane composition according to step (j) of FIG. 3 for the treatment of Peyronie's disease.

[0072] [Figure 10] FIG. 10 is a graph showing the concentration of transthyretin in human amniotic fluid filtrate (step (e') and / or step (f') of FIG. 2) prior to mixing with the micronized human amniotic membrane composition of step (i) of FIG. 3 for the treatment of Peyronie's disease.

[0073] [Figure 11] FIG. 11 is a graph showing the total concentration of transthyretin in a multi-part processed human amniotic membrane composition according to step (j) of FIG. 3 for the treatment of Peyronie's disease.

[0074] [Figure 12] FIG. 12 is a graph showing the total concentration of exosomes in a multi-part processed human amniotic membrane composition according to step (j) of FIG. 3 for the treatment of Peyronie's disease.

[0075] [Figure 13]Figures 13A and 13B show microscopic images with DAPI staining of the multi-part processed human amniotic membrane composition using a 50 micron scale, Figures 13C and 13D show microscopic images with DAPI staining of the multi-part processed human amniotic membrane composition using a 20 micron scale, and Figures 13E and 13F show microscopic images with DAPI staining of the multi-part processed human amniotic membrane composition using a 10 micron scale.

[0076] [Figure 14] FIG. 14 shows exemplary endogenous cellular pathways activated by a multi-part processed human amniotic membrane composition for the treatment of Peyronie's Disease. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0077] The present invention will now be described more fully hereinafter with reference to the accompanying drawings, in which exemplary embodiments of the invention are shown, however, this invention may be embodied in many different forms and should not be construed as limited to the representative embodiments set forth herein.

[0078] The exemplary embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention, and will enable any person skilled in the art to make, use and practice the invention. Like reference numerals refer to like elements throughout the various drawings. Furthermore, in this specification and in the claims that follow, reference will be made to a number of terms that are defined to have the following meanings:

[0079] It must 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.

[0080] Concentrations, amounts, and other numerical data may be expressed or presented in a range format herein. It should be understood that such range formats are used merely for convenience and brevity, and thus should be interpreted flexibly to include not only the numerical values ​​explicitly set forth as the limits of the range, but also all individual numerical values ​​and subranges encompassed within the range, as if those numerical values ​​or subranges were explicitly set forth. As an example, a numerical range of "about 1 to 5" should be interpreted as including not only the value explicitly set forth as "about 1 to 5", but also the individual values ​​and subranges within the stated range. Thus, this numerical range includes not only 1, 2, 3, 4, and 5, but also individual values ​​such as 2, 3, and 4, and subranges such as 1 to 3, 2 to 4, and 3 to 5. The same principle applies to ranges that set forth only one numerical value as a minimum or maximum value. Moreover, such interpretation should be applied regardless of the breadth of the range or the properties described.

[0081] 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.

[0082] The medical benefits of human placental membranes have become increasingly evident over the past few years, resulting in the development of a variety of membrane combinations for applications ranging from cosmetic to invasive surgical procedures.

[0083] With the above generally favorable medical benefits of human placental membrane in mind, and considering the endogenous components contained in human amniotic membrane and human amniotic fluid, it was believed that a therapeutic multi-part processed human amniotic membrane composition would be useful in treating Peyronie's disease, to reduce symptoms associated with Peyronie's disease, reduce the size of plaques associated with Peyronie's disease, improve penile function in subjects suffering from Peyronie's disease, and restore penile function in subjects suffering from Peyronie's disease. Generally, human amniotic fluid (also referred to herein as "amniotic fluid" or "AF") is a clear, slightly yellowish liquid that surrounds the fetus during pregnancy. AF contains nutrients and growth factors that promote fetal growth, and provides mechanical cushioning and antimicrobial factors that protect the fetus. In addition, AF contains proteins, electrolytes, immunoglobulins, and vitamins therein. Furthermore, AF contains high levels of sulfated glycosaminoglycans, IGFBP-1, and hepatocyte growth factor (HGF). Amniotic fluid and amniotic membrane also contain transthyretin.

[0084] The human amniotic membrane (also referred to herein as "amniotic membrane") has a thickness of 0.02-0.05 mm. The human amniotic membrane has no blood vessels, nerves, or lymphatics and receives nutrition by diffusion in the uterus. The amniotic membrane is in direct contact with the amniotic fluid. The human amniotic membrane has three layers: an epithelial layer, a thick basement membrane, and avascular mesenchymal tissue. These contain types I and III-VII collagens, along with fibronectin and laminin, and high concentrations of proteoglycans and glycoproteins. The functions of the amniotic membrane include physical protection of the fetus, protection from bacterial infection, regulation of pH, and secretion of growth factors and other molecules. These perform antibacterial and anti-inflammatory functions. In addition, the applicants have noticed that the amniotic membrane contains high levels of hyaluronic acid (HA) and IL-1ra.

[0085] In terms of penile defects (e.g., Peyronie's disease) and resolution of penile defects, the amniotic membrane provides stromal support, ECM repair, and anti-inflammatory factors. In terms of penile defects, AF provides hydration, stromal support, and matrix remodeling factors. Amniotic fluid also contains transthyretin.

[0086] While human amniotic membrane and human amniotic fluid have little medical and / or therapeutic use in their natural state, when the endogenous components contained therein are further processed into pharmaceutical products containing the above components in appropriate concentrations and amounts, they can be used (by internal injection / administration) to treat Peyronie's disease by reducing plaque size, promoting endogenous remodeling of the corpus cavernosum, improving the ECM properties of the corpus cavernosum, reducing inflammation within the corpus cavernosum and other penile tissues, and improving / restoring the function of other penile tissues, thereby restoring normal sexual function. Multi-part processed amniotic membrane composition

[0087] With the above in mind, disclosed herein are multi-part processed amniotic membrane compositions derived from human amniotic membrane and amniotic fluid, where the components when mixed together mimic, contain, and / or retain a cellular and / or extracellular profile resembling the endogenous profile of human amniotic membrane and / or amniotic fluid, e.g., in vivo, particularly in comparison to their isolates. Also, to further maximize the retention of the cellular and / or extracellular profile resembling the endogenous profile of human amniotic membrane and / or amniotic fluid, e.g., in vivo, these compositions are not treated with exogenous enzymes during manufacture. Moreover, these compositions do not include added enzymes, such as collagenase, in the final product (i.e., multi-part processed amniotic membrane composition), thereby advantageously minimizing and / or eliminating immunogenic responses and / or other side effects seen with conventional Peyronie's disease treatments.

[0088] In particular, the disclosed compositions are prepared using fresh human amniotic membrane and amniotic fluid (collected and processed within 0-72 hours after removal from the human subject), preferably maintained at 4°C-8°C during collection and processing to prevent and / or reduce endogenous enzymatic degradation processes during manufacture of the multi-part processed amniotic membrane compositions. Similarly, the disclosed multi-part processed amniotic membrane compositions are subsequently stored at temperatures between 4°C-8°C to maintain a preferred shelf life and minimize degradation of these compositions during storage.

[0089] Unlike compositions of the prior art, the disclosed compositions advantageously do not undergo biochemical and / or enzymatic digestion, resulting in compositions that contain and / or retain a majority of the cellular and / or extracellular profile (when compared to the endogenous profile of human umbilical cord in vivo). Also, due to the ease of preparation (sterile and / or antiseptic preparation) and convenience (e.g., preparation and use at the point of use within a urologist's office or other medical facility) of these compositions, as well as the non-immunogenic properties of these compositions, these compositions can be used in the human body (e.g., as injectable compositions) to advantageously treat Peyronie's disease and / or reduce the size of plaques in people affected by Peyronie's disease.

[0090] The multi-part processed human amniotic membrane composition is configured to treat Peyronie's disease by injecting an effective amount of the multi-part processed human amniotic membrane composition into a subject in need of treatment. The multi-part processed human amniotic membrane composition specifically includes (a) a micronized human amniotic membrane composition and (b) an aqueous human amniotic fluid filtrate configured to resuspend the micronized human amniotic membrane composition, and the multi-part processed human amniotic membrane composition is not treated with exogenous enzymes during its manufacture and does not include exogenous enzymes added thereto. The elimination of exogenous enzymes during the manufacture of the composition can avoid undesirable enzymatic degradation / digestion in the micronized human amniotic membrane composition and the aqueous human amniotic fluid filtrate in the multi-part processed human amniotic membrane composition, and further ensures that these compositions have an improved endogenous cellular and extracellular profile (a profile similar to that of human amniotic membrane and amniotic fluid in vivo), especially compared to conventional compositions using amniotic membrane and amniotic fluid and / or cell isolates derived therefrom.

[0091] In certain embodiments, the micronized human amniotic membrane composition is a dried and / or pulverized (e.g., freeze-pulverized) human amniotic membrane that has not been subjected to exogenous enzymatic digestion during its preparation, and preserves and / or inhibits degradation of endogenous hyaluronic acid (HA) and / or hyaluronan, fibronectin, insulin growth factor binding protein-1 (IGFBP-1), sulfated glycosaminoglycans (sGAGs), exosomes, interleukin-1 receptor antagonist (IL-1ra), hepatocyte growth factor (HGF), transthyretin, or any combination thereof. For reasons further described below, these components advantageously serve to reduce the size of plaques associated with Peyronie's disease and / or further promote endogenous remodeling of the corpus cavernosum and other penile tissues to restore normal sexual function.

[0092] The micronized human amniotic membrane composition of the multi-part processed human amniotic membrane composition is either polydisperse or monodisperse, with particle diameter sizes of greater than 1 μm to less than 500 μm, greater than 1 μm to less than 300 μm, greater than 1 μm to less than 100 μm, greater than 1 μm to less than 50 μm, or greater than 1 μm to less than 25 μm, such that the micronized human amniotic membrane composition is sufficiently mixed, wetted, reconstituted, dispersed, and / or suspended when mixed with aqueous human amniotic fluid filtrate. It is further contemplated that any endpoint within the stated ranges may also be an endpoint of an additional range. The micronized human amniotic membrane composition preferably has particle diameters within the above-mentioned ranges so that uniform mixing, dispersion, wetting, and suspension occurs in the multi-part processed human amniotic membrane composition (when the micronized human amniotic membrane composition is mixed with the aqueous human amniotic fluid filtrate to form the multi-part processed human amniotic membrane composition), thereby providing excellent bioavailability of the hyaluronic acid and / or hyaluronan, fibronectin, insulin growth factor binding protein-1 (IGFBP-1), sulfated glycosaminoglycans (sGAGs), exosomes, interleukin-1 receptor antagonist (IL-1ra), hepatocyte growth factor (HGF), and transthyretin in the multi-part processed human amniotic membrane composition, and minimizing side effects and / or discomfort associated with injectable compositions, resulting in high therapeutic efficacy when administered to a subject in need of treatment.

[0093] In certain embodiments, particle diameters of the micronized human amniotic membrane composition exceeding 500 μm can result in poor mixing, poor wetting, non-uniform dispersion, and / or poor suspension, ultimately resulting in either an unusable and / or suboptimal composition having insufficient bioavailability of the hyaluronic acid (HA) and / or hyaluronan, fibronectin, insulin growth factor binding protein-1 (IGFBP-1), sulfated glycosaminoglycans (sGAGs), exosomes, interleukin-1 receptor antagonist (IL-1ra), hepatocyte growth factor (HGF), and transthyretin in the micronized human amniotic membrane composition (having a diameter greater than 500 μm), resulting in less effective treatment of Peyronie's disease and / or more side effects and / or more discomfort upon administration.

[0094] As mentioned above, the multi-part processed human amniotic membrane composition further comprises an aqueous human amniotic fluid filtrate configured to return and / or suspend the micronized human amniotic membrane composition. The aqueous human amniotic fluid filtrate is preferably prepared from the same human donor as the amniotic membrane (and the micronized human amniotic membrane composition (described above)) through one or more separation steps (e.g., a filtration step, which is described further below). In certain embodiments, the aqueous human amniotic fluid filtrate comprises hyaluronic acid (HA), and / or hyaluronan, fibronectin, insulin growth factor binding protein-1 (IGFBP-1), sulfated glycosaminoglycan (sGAG), exosomes, interleukin-1 receptor antagonist (IL-1ra), hepatocyte growth factor (HGF), transthyretin, or a combination thereof, in a concentration sufficient to treat Peyronie's disease and / or reduce the size of Peyronie's disease plaques in the multi-part processed human amniotic membrane composition. In certain alternative embodiments, the aqueous human amniotic fluid filtrate may be further diluted prior to inclusion in the multi-part processed human amniotic fluid composition to obtain a sufficient concentration of hyaluronic acid (HA), and / or hyaluronan, fibronectin, insulin growth factor binding protein-1 (IGFBP-1), sulfated glycosaminoglycans (sGAGs), exosomes, interleukin-1 receptor antagonist (IL-1ra), hepatocyte growth factor (HGF), transthyretin, or combinations thereof in the multi-part processed human amniotic membrane composition to treat Peyronie's disease and / or reduce the size of Peyronie's disease plaques. If diluted, the aqueous human amniotic fluid filtrate is diluted with an isotonic solution such as phosphate buffered saline (or any one of lactated Ringer's (pH 6.5, 6 g / L NaCl, 3.1 g / L sodium lactate, 0.3 g / L KCl, and 0.2 g / L CaCl), isotonic saline (0.9 wt% NaCl), plasmalyte® (pH 7.4, 5.26 g / L NaCl, 0.37 g / L KCl, 0.30 g / L magnesium chloride hexahydrate, 3.68 g / L sodium acetate trihydrate, 5.02 g / L sodium gluconate)).

[0095] FIG. 4 is a photograph of a multi-part processed human amniotic membrane composition in an exemplary sterile container (100) stored at −20° C. to −80° C. and ready for subsequent use. Other sterile containers include ampoules, pre-filled syringes, and / or vials, also stored at −20° C. to −80° C. and ready for subsequent use. FIG. 13A and FIG. 13B show microscopic images with DAPI staining of the multi-part processed human amniotic membrane composition using a 50 micron scale, FIG. 13C and FIG. 13D show microscopic images with DAPI staining of the multi-part processed human amniotic membrane composition using a 20 micron scale, and FIG. 13E and FIG. 13F show microscopic images with DAPI staining of the multi-part processed human amniotic membrane composition using a 10 micron scale. Also, Figures 5-9, 11 and 12 show typical concentration profiles of HA, SGAG, IL-1RA, HGF, IGF-BP1, transthyretin, and exosomes contained in a multi-part processed human amniotic membrane composition (i.e., a composition having a micronized human amniotic membrane composition and an aqueous human amniotic fluid filtrate) configured to treat Peyronie's disease by internal injection into a subject in need of treatment. Meanwhile, in contrast to Figure 11, Figure 10 shows transthyretin contained only in the aqueous human amniotic fluid filtrate (i.e., excluding the micronized human amniotic membrane composition). In one embodiment, as shown in Figures 5-9, 11 and 12, the multi-part processed human amniotic membrane composition has a concentration profile of 1.2 x 10 6 pg / mL ~ 1.1 × 10 8 pg / mL, and / or 1.3 × 10 6 pg / mL~5.5×10 7 HA at a concentration of 2.5 x 10 pg / mL 6 pg / mL~9×10 6 pg / mL, and / or 2.75 × 10 6 pg / mL~5×10 6 pg / mL IGFBP-1, 5 × 10 7 pg / mL ~ 8.0 × 10 7 pg / mL, and / or 5.3 × 107 pg / mL ~ 7.5 × 10 7 pg / mL of sGAG, particle diameter of 50-200 nm, and 1.0 × 10 9 pg / mL~2.0×10 9 pg / mL, and / or 1.2 × 10 9 pg / mL ~ 1.7 × 10 9 Exosomes at a concentration of pg / mL, 2 × 10 4 pg / mL~1.5×10 5 pg / mL, and / or 2.1 × 10 4 pg / mL~7×10 4 pg / mL IL-1ra, 900 pg / mL to 3000 pg / mL, and / or 1000 pg / mL to 1500 pg / mL HGF, 1.25 × 10 4 ~2.25×10 4 pg / mL, and / or 1.3 × 10 4 ~2.15×10 4 pg / mL transthyretin, or any combination thereof. Meanwhile, in certain embodiments, the multi-part processed human amniotic membrane composition comprises 1.2×10 pg / mL transthyretin when the micronized human amniotic membrane composition is reconstituted and suspended in aqueous human amniotic fluid filtrate. 6 pg / mL ~ 1.1 × 10 8 pg / m, L and / or 1.3 × 10 6 pg / mL~5.5×10 7 HA at a concentration of pg / mL and 2.5 × 10 6 pg / mL~9×10 6 pg / mL, and / or 2.75 × 10 6 pg / mL~5×10 6 pg / mL IGFBP-1 and 5 × 10 7 pg / mL ~ 8.0 × 10 7 pg / mL, and / or 5.3 × 10 7 pg / mL ~ 7.5 × 10 7 pg / mL of sGAG and a particle diameter of 50-200 nm, with a concentration of 1.0 × 10 9 pg / mL~2.0×10 9 pg / mL, and / or 1.2 × 10 9 pg / mL ~ 1.7 × 10 9pg / mL concentration of exosomes and 2 × 10 4 pg / mL~1.5×10 5 pg / mL, and / or 2.1 × 10 4 pg / mL~7×10 4 pg / mL IL-1ra, 900pg / mL to 3000pg / mL, and / or 1000pg / mL to 1500pg / mL HGF, and 1.25 × 10 4 ~2.25×10 4 pg / mL, and / or 1.3 × 10 4 ~2.15×10 4 pg / mL transthyretin. In certain embodiments, both the micronized human amniotic membrane composition and the aqueous human amniotic fluid filtrate are sterile and / or preservative, and the multi-part processed human amniotic membrane composition is similarly sterile and / or preservative. In certain embodiments, both the micronized human amniotic membrane composition and the aqueous human amniotic fluid filtrate are non-immunogenic, and the multi-part processed human amniotic membrane composition is similarly non-immunogenic. In certain embodiments, treatment (i.e., internal injection / administration to a subject in need of treatment) of the multi-part processed human amniotic membrane composition treats Peyronie's disease, reduces the size of Peyronie's disease plaques, reduces symptoms associated with Peyronie's disease, improves penile function, particularly aiding in pain-free erections, and / or restores penile function, including pain-free erections.

[0096] In view of the above, and without wishing to be bound by theory, transthyretin is the primary active ingredient / therapeutic agent for reducing the size of plaques associated with Peyronie's disease, treating Peyronie's disease, alleviating symptoms of Peyronie's disease, and / or restoring and / or improving penile function in people suffering from Peyronie's disease. Transthyretin is present in the multi-part processed human amniotic membrane composition in an effective amount for treating Peyronie's disease, alleviating symptoms associated with Peyronie's disease, and / or reducing the size of plaques in Peyronie's disease, and in certain embodiments, transthyretin is present in the multi-part processed human amniotic membrane composition at a concentration of 10-50 pg / mL. Although the mechanism of action has yet to be elucidated, transthyretin has previously been found to bind to beta amyloid protein (associated with the early stages of Alzheimer's) and block the natural tendency of beta amyloid to accumulate as plaques. It is believed that the mechanism of action for reducing the size of plaques associated with Peyronie's disease (and treating Peyronie's disease, alleviating the symptoms of Peyronie's disease, and / or restoring and / or improving penile function in people with Peyronie's disease) may also be similar.

[0097] In addition to the transthyretin contained in the composition described above, the multi-part processed human amniotic membrane composition also contains 1.2×10 6 pg / mL ~ 1.1 × 10 8 pg / mL, and / or 1.3 × 10 6 pg / mL~5.5×10 7 HA at a concentration of pg / mL and 2.5 × 10 6 pg / mL~9×10 6 pg / mL, and / or 2.75 × 10 6 pg / mL~5×10 6 pg / mL IGFBP-1 and 5 × 10 7 pg / mL ~ 8.0 × 10 7 pg / mL, and / or 5.3 × 10 7 pg / mL ~ 7.5 × 10 7pg / mL of sGAG, particle diameter of 50-200 nm, and 1.0 × 10 9 pg / mL~2.0×10 9 pg / mL, and / or 1.2 × 10 9 pg / mL ~ 1.7 × 10 9 pg / mL concentration of exosomes and 2 × 10 4 pg / mL~1.5×10 5 pg / m, L and / or 2.1 × 10 4 pg / mL~7×10 4 pg / mL IL-1ra, 900pg / mL to 3000pg / mL, and / or 1000pg / mL to 1500pg / mL HGF, and 1.25 × 10 4 ~2.25×10 4 pg / mL, and / or 1.3 × 10 4 ~2.15×10 4pg / mL transthyretin further plays a role in the remodeling and recovery function of penile tissue, helping to treat and improve penile function in people suffering from Peyronie's disease. In particular, Figure 14 shows a schematic of the endogenous mechanisms of action of IL-1RA, IGF-BP1, HGF, HA and sGAG. Hyaluronic acid (HA) is a major component of the extracellular matrix [Toole 2001]. HA is a non-sulfated glycosaminoglycan and provides the backbone of sulfated glycosaminoglycans among others. HA also binds to integrins to stabilize ECM and absorb water, another major component of ECM. The large molecular weight of HA results in unique biophysical properties such as high viscoelasticity and high colloid osmotic pressure [Laurent 1995][Cowman and Matsuoka 2005]. In addition, HA leads to stabilization of the extracellular matrix, water retention, and control of protein distribution. Sulfated glycosaminoglycans (sGAGs) interact with other components of the ECM to organize and form a hydrophilic matrix suitable for remodeling [Couchman 2012][Prydz 2000]. sGAGs also bind to HA backbones and deliver factors to tissues. IGFBP-1 is the predominant IGF-binding protein in amniotic fluid, fetal, and maternal circulation [Hills 1996][Hills 1996]. IGFBP1 binds IGFs, extending their half-life and altering their interactions with cell surface receptors. IL-1ra binds nonproductively to IL-1 (inflammatory cytokine) receptors, preventing IL-1 (an inflammatory molecule) from signaling. This alters a variety of IL-1-associated immune and inflammatory responses. HGF is a cell proliferation, motility, and morphogenesis factor secreted by mesenchymal cells. HGF has been shown to play a key role in embryonic organ development, particularly myogenesis, adult organ regeneration, and wound healing. HGF has been associated with enhanced scar-free wound healing capabilities of fibroblasts derived from oral mucosal tissue [Dally 2017], and HGF at concentrations disclosed herein may play a role in reducing the size of Peyronie's plaques (in addition to the therapeutic effects of transthyretin and the plaque reduction function). Method for producing a multi-part processed amniotic membrane composition

[0098] Figures 1-3 provide, respectively, a schematic diagram of the steps involved in producing a micronized human amniotic membrane composition, a schematic diagram of the steps involved in producing an aqueous human amniotic fluid filtrate, and a schematic diagram of the steps involved in producing a multi-part processed human amniotic membrane composition by mixing the micronized human amniotic membrane composition with and / or suspending the micronized human amniotic membrane composition in the aqueous human amniotic fluid filtrate. More specifically, Figure 1 is a schematic diagram of the steps involved in producing a micronized human amniotic membrane composition of a multi-part processed amniotic membrane composition for the treatment of Peyronie's disease. FIG. 2 is a schematic diagram of the steps involved in producing the human amniotic fluid filtrate contained in a multi-part processed human amniotic membrane composition for the treatment of Peyronie's disease, and FIG. 3 is a schematic diagram of the steps involved in mixing the micronized human amniotic membrane composition of FIG. 1 with the amniotic fluid filtrate of FIG. 2 to produce a multi-part processed human amniotic membrane composition having a desired concentration profile of HA, sGAG, IGFBP-A, IL-1ra, HGF, and transthyretin in a predetermined amount for internal administration to a subject in need of treatment and for the treatment of Peyronie's disease.

[0099] With specific reference to Figure 1, steps are disclosed for manufacturing / preparing a micronized human amniotic membrane composition having a desired particle size / diameter. However, to ensure that the amniotic tissue is healthy / disease-free and to further minimize risk during preparation and subsequent end use of the multi-part processed human amniotic membrane composition for treating Peyronie's disease, prior to step (a), the human amniotic membrane (and human amniotic fluid) is tested for transmissible diseases. After the human amniotic membrane tissue (and human amniotic fluid) is confirmed to be healthy / disease-free, the human amniotic membrane is maintained at a temperature of 4°C to 8°C prior to processing the amniotic membrane, as shown in steps (a)-(g) of Figure 1. As shown in FIG. 1, step (a) involves preparing fresh human amniotic membrane, and step (a') involves preparing fresh human amniotic fluid (preferably from the same donor) within 24 to 96 hours after extraction from the human subject, more preferably within 24 to 72 hours after extraction from the human subject, to ensure the freshness of the human amniotic fluid as well as the human amniotic membrane (i.e., the tissue and the cells that constitute that tissue) and minimize degradation (enzymatic degradation) due to necroptosis and / or apoptosis.

[0100] In step (b), the human amniotic membrane is separated from other human placental components, thereby producing an isolated human amniotic membrane, which may be resized to a desired size in this step prior to proceeding to step (c). For example, in certain embodiments, it may be preferable to resize the isolated human amniotic membrane to a portion size of 15-80 grams, more preferably a portion size of 30-60 grams, for ease of handling and processing in subsequent steps.

[0101] After step (b), in step (c), the isolated human amniotic membrane is rinsed with an alcohol solution to wash away irrelevant non-amniotic human placental components and / or other potential contaminants. In certain aspects, 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 a most preferred embodiment, the alcohol is 70% isopropyl alcohol. Isopropyl alcohol is advantageously preferred over other commercially available laboratory and / or pharmaceutical grade alcohols, such as ethanol, because it disinfects and cleans the human amniotic membrane without damaging the amniotic tissue (e.g., excessively dehydrating, causing apoptotic and / or necrotic processes).

[0102] After step (c), in step (d), the rinsed human amniotic membrane of step (c) is washed with an isotonic solution. In particular, step (d) comprises placing one or more portions of human amniotic membrane (15-80 grams) into a container with a predetermined amount (e.g., 300 mL-1000 mL, preferably 500 mL) of isotonic solution, which is preferably phosphate buffered saline (PBS) (i.e., 1×PBS) (alternatively, any one of lactated Ringer's (pH 7.4, 6 g / L NaCl, 3.1 g / L sodium lactate, 0.3 g / L KCl, and 0.2 g / L CaCl), isotonic saline (0.9 wt% NaCl), plasmalyte® (pH 7.4, 5.26 g / L NaCl, 0.37 g / L KCl, 0.30 g / L magnesium chloride hexahydrate, 3.68 g / L sodium acetate trihydrate, 5.02 g / L sodium gluconate)). The human amniotic membrane portion is washed by placing the container on a stirring plate, placing a stir bar in the container (containing PBS and the human amniotic membrane portion), and stirring (medium to high speed) the human amniotic membrane portion in the isotonic solution for 5 to 15 minutes. In one embodiment, washing step (d) is repeated 1 to 5 times by decanting the "used" isotonic solution and pouring a predetermined amount (e.g., 300 mL to 1000 mL, preferably 500 mL) of new isotonic solution into the container and washing the one or more human amniotic membrane portions again.

[0103] As further shown in Figure 1, before step (b), during step (b), before step (c), during step (d), before step (d), or during step (d), the presence of blood clots and / or blood pools within the human amniotic membrane and / or amniotic membrane portions is further determined, and if present, these blood clots are removed using suction or other mechanical removal means (e.g., scalpel and forceps) to minimize immunogenic components (e.g., hemoglobin and / or heme-related components from the human amniotic membrane donor) in the final micronized human amniotic membrane composition and the final multi-part processed human amniotic membrane composition. These washing steps are essential to maintain an antiseptic and / or sterile working environment to prevent and / or reduce the introduction of any contaminants during the manufacture of the micronized human amniotic membrane composition and the final multi-part processed human amniotic membrane composition.

[0104] After step (d), in step (e), the washed human amniotic membrane of step (d) is dried to produce a dried human amniotic membrane. In particular, in step (e), the amniotic membrane is laid flat in a biosafety cabinet with a circulating fan at ambient temperature for 2 to 6 hours or in a dehydrator (at a temperature of 30° C. to 40° C.) for 15 minutes to 24 hours.

[0105] After step (e), the dried human amniotic membrane is reduced to a smaller portion size and / or subjected to a grinding process. In particular, in step (f), the dried human amniotic membrane is subjected to a grinding process and / or a freeze-grinding process configured to obtain particles (polydisperse or monodisperse particles) having a size of more than 1 μm to 500 μm, more than 1 μm to 400 μm, more preferably 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 400 μm, more than 50 μm to 300 μm, more than 50 μm to 200 μm, where any of the endpoints of these ranges may be endpoints of further ranges. In certain embodiments, step (f) is a freeze-grinding process (e.g., as described in US 20160287749, US 20170203004, and US Patent No. 10,105,398, which are incorporated by reference in their entireties) in which the dried human amniotic membrane of step (e) is placed into a liquid nitrogen-cooled cryomill chamber and ground therein, thereby producing a micronized human amniotic membrane composition having the particle diameters described above. In certain embodiments, the duration of each grinding and / or milling (cryomill) cycle is 1-4 minutes, 1-3 minutes, 1.5-2.5 minutes. As further shown in Figure 1, the method of producing a micronized human amniotic membrane composition may further include step (g) in which the grinding and / or milling process of step (f) is repeated for a predetermined number of additional cycles (e.g., 1 cycle, 2 cycles, 3 cycles, 4 cycles, 5 cycles, 6 cycles, etc.), each additional cycle having a predetermined duration (e.g., 1-4 minutes, 1-3 minutes, 1.5-2.5 minutes, etc.). These additional cycles result in smaller and more uniform particle diameters of the micronized human amniotic membrane composition.As mentioned above, the micronized human amniotic membrane composition of step (f) and / or step (g), when mixed and / or resuspended in aqueous human amniotic fluid filtrate, comprises an effective amount / concentration of hyaluronic acid (HA) and / or hyaluronan, fibronectin, insulin growth factor binding protein-1 (IGFBP-1), sulfated glycosaminoglycans (sGAGs), exosomes, interleukin-1 receptor antagonist (IL-1ra), hepatocyte growth factor (HGF), transthyretin, or any combination thereof, thereby constituting a multi-part processed amniotic membrane composition for treating Peyronie's disease, alleviating symptoms associated with Peyronie's disease, reducing plaque size associated with Peyronie's disease, and / or improving and / or restoring penile function in a subject in need of treatment.

[0106] Each of steps (a)-(g) is preferably performed under a sterile and / or aseptic environment to result in a sterile and non-immunogenic micronized human amniotic membrane composition. Each of steps (a)-(h) is performed below 21° C. to prevent and / or reduce endogenous enzymatic degradation processes during the manufacture of the micronized human amniotic membrane composition, while maintaining the desired profile of hyaluronic acid (HA) and / or hyaluronan, fibronectin, insulin growth factor binding protein-1 (IGFBP-1), sulfated glycosaminoglycans (sGAGs), exosomes, interleukin-1 receptor antagonist (IL-1ra), hepatocyte growth factor (HGF), and / or transthyretin.

[0107] After step (f) or step (g), the micronized human amniotic membrane composition may optionally be placed into a first sterile container and sealed for subsequent use / mixing and / or suspension with the aqueous human amniotic fluid filtrate (step (h)). Alternatively, step (h) may be omitted and the micronized human amniotic membrane composition may be immediately mixed and / or suspended in the aqueous human amniotic fluid filtrate, as shown in step (i) of FIG. 3.

[0108] Figure 2 is a schematic diagram of the steps involved in producing human amniotic fluid filtrate for inclusion in a multi-part processed human amniotic membrane composition for the treatment of Peyronie's disease. The steps shown in Figure 2 may be performed before, during, or after steps (a)-(h) of Figure 1. As shown in Figure 2, in step (a'), fresh human amniotic fluid is prepared from a healthy / non-diseased donor within 0-72 hours of extraction / removal from the donor and maintained at a temperature of 4°C-8°C.

[0109] Next, in step (b'), the fresh human amniotic fluid is filtered through a 200 micron filter to produce a first human amniotic fluid retentate (first retentate) and a first human amniotic fluid filtrate. The first retentate is then discarded. Next, in step (c'), the first human amniotic fluid filtrate is filtered through a 100 micron filter to produce a second human amniotic fluid retentate (second retentate) and a second human amniotic fluid filtrate. The second retentate is then discarded. Next, in step (d'), the second human amniotic fluid filtrate is filtered through a 70 micron filter to produce a third human amniotic fluid retentate (third retentate) and a third human amniotic fluid filtrate, where the third human amniotic fluid filtrate is acellular, contains only particles smaller than 70 microns, and is sterile. The third human amniotic fluid filtrate comprises hyaluronic acid (HA) and / or hyaluronan, fibronectin, insulin growth factor binding protein-1 (IGFBP-1), sulfated glycosaminoglycans (sGAGs), exosomes, interleukin-1 receptor antagonist (IL-1ra), hepatocyte growth factor (HGF), transthyretin, or combinations thereof.

[0110] The third human amniotic fluid filtrate then optionally undergoes step (f'), in which the third human amniotic fluid filtrate is diluted with a predetermined amount of balanced salt solution / isotonic solution (e.g., phosphate buffered saline (PBS) (i.e., 1xPBS) (alternatively, one of lactated Ringer's (pH 6.5, 6g / L NaCl, 3.1g / L sodium lactate, 0.3g / L KCl, 0.2g / L CaCl), isotonic saline (0.9wt% NaCl), plasmalyte® (pH 7.4, 5.26g / L NaCl, 0.37g / L KCl, 0.30g / L magnesium chloride hexahydrate, 3.68g / L sodium acetate trihydrate, 5.02g / L sodium gluconate)) to produce a diluted third human amniotic fluid filtrate. This step is performed if the concentrations of hyaluronic acid (HA) and / or hyaluronan, fibronectin, insulin growth factor binding protein-1 (IGFBP-1), sulfated glycosaminoglycans (sGAGs), exosomes, interleukin-1 receptor antagonist (IL-1ra), hepatocyte growth factor (HGF), and / or transthyretin are too high (too concentrated) to obtain the concentration profile of the multi-part processed amniotic membrane composition shown in step (i) of Figure 3.

[0111] Each of steps (a') through (f') is performed below 21° C. to prevent and / or reduce endogenous enzymatic degradation processes during the preparation of the filtrates disclosed herein. These steps are performed in a sterile and / or preserved environment, and as noted above, the third human amniotic fluid filtrate (step (e')) and / or the diluted third human amniotic fluid filtrate (step (f')) are sterile, preserved, and / or non-immunogenic.

[0112] After step (e') or step (f'), the micronized human amniotic membrane composition (produced in either step (f) or step (g) of FIG. 1) is mixed with either a third human amniotic fluid retentate or a diluted third human amniotic fluid filtrate, thereby producing a multi-part processed human amniotic membrane composition configured to treat Peyronie's disease by internal injection, neither step including the introduction of an exogenous enzyme. In one embodiment, as shown in step (i) of FIG. 3 and FIGS. 5-9, 11 and 12, the multi-part processed human amniotic membrane composition is prepared by mixing the micronized human amniotic membrane composition with 1.2×10 6 pg / mL ~ 1.1 × 10 8 pg / m, L and / or 1.3 × 10 6 pg / mL~5.5×10 7 HA at a concentration of 2.5 x 10 pg / mL 6 pg / mL~9×10 6 pg / mL, and / or 2.75 × 10 6 pg / mL~5×10 6 pg / mL IGFBP-1, 5 × 10 7 pg / mL ~ 8.0 × 10 7 pg / mL, and / or 5.3 × 10 7 pg / mL ~ 7.5 × 10 7 pg / mL of sGAG, particle diameter of 50-200 nm, and 1.0 × 10 9 pg / mL~2.0×10 9 pg / mL, and / or 1.2 × 10 9 pg / mL ~ 1.7 × 10 9 Exosomes at a concentration of pg / mL, 2 × 10 4 pg / mL~1.5×10 5 pg / mL, and / or 2.1 × 10 4 pg / mL~7×10 4 pg / mL IL-1ra, 900 pg / mL to 3000 pg / mL, and / or 1000 pg / mL to 1500 pg / mL HGF, 1.25 × 10 4 ~2.25×104 pg / mL and / or 1.3×10 4 ~2.15×104 pg / mL transthyretin, or any combination thereof, while in certain embodiments, the multi-part processed human amniotic membrane composition comprises 1.2×10 pg / mL transthyretin when the micronized human amniotic membrane composition is reconstituted, dispersed, and / or suspended in aqueous human amniotic fluid filtrate. 6 pg / mL ~ 1.1 × 10 8 pg / mL, and / or 1.3 × 10 6 pg / mL~5.5×10 7 HA at a concentration of pg / mL and 2.5 × 10 6 pg / mL~9×10 6 pg / m, L and / or 2.75 × 10 6 pg / mL~5×10 6 pg / mL IGFBP-1 and 5 × 10 7 pg / mL ~ 8.0 × 10 7 pg / mL, and / or 5.3 × 10 7 pg / mL ~ 7.5 × 10 7 pg / mL of sGAG and a particle diameter of 50-200 nm, with a concentration of 1.0 × 10 9 pg / mL~2.0×10 9 pg / m, L and / or 1.2 × 10 9 pg / mL ~ 1.7 × 10 9 pg / mL concentration of exosomes and 2 × 10 4 pg / mL~1.5×10 5 pg / mL, and / or 2.1 × 10 4 pg / mL~7×10 4 pg / mL IL-1ra, 900pg / mL to 3000pg / mL, and / or 1000pg / mL to 1500pg / mL HGF, and 1.25 × 10 4 ~2.25×10 4 pg / mL, and / or 1.3 × 10 4 ~2.15×10 4 and transthyretin in pg / mL.

[0113] As further shown in step (j) of Figure 3, and as further described below with respect to the kits disclosed herein, the multi-part processed amniotic membrane composition for treating Peyronie's disease is then placed into a sterile container (e.g., a sterile vial, a sterile ampoule, or a sterile pre-filled syringe) for use by internal injection into the penis (capsule, corpus cavernosum, and / or plaque) to treat Peyronie's disease and / or minimize symptoms of Peyronie's disease. To minimize and / or prevent undesirable side effects and / or to minimize immunogenic responses in a subject upon treatment with the disclosed compositions, the multi-part processed amniotic membrane composition for treating Peyronie's disease that is placed and / or stored in a sterile container is also sterile, preserved, and / or free of contaminants. Kits Containing Multi-Part Treated Amniotic Membrane Compositions

[0114] Also disclosed herein is a kit including a multi-part processed human amniotic membrane composition configured to treat Peyronie's Disease by internal injection of an effective amount into a subject in need of treatment. The kit includes a sterile container in which a multi-part processed human amniotic membrane composition configured to treat Peyronie's Disease by internal injection is stored, the multi-part processed human amniotic membrane composition configured to treat Peyronie's Disease by internal injection includes a micronized human amniotic membrane composition suspended in aqueous human amniotic fluid filtrate, but does not include added exogenous enzymes. For example, FIG. 4 is a photograph of a multi-part processed human amniotic membrane composition contained within an exemplary sterile container (100) stored at -20°C to -80°C and ready for subsequent use. Other exemplary sterile containers include ampoules, pre-filled syringes, and / or vials, also stored at -20°C to -80°C and ready for subsequent use. The kits disclosed herein, more particularly the containers, may be configured for single use (e.g., pre-filled syringes or ampoules) or multiple use (e.g., vials) and may contain a predetermined volume of 0.5 mL to 5 mL, 1 mL to 4 mL, or 1.5 to 3 mL, and any endpoint within these ranges may be an endpoint of an additional range. In certain embodiments, single-use kits may contain the volumes disclosed above (e.g., 0.5 mL, 0.75 mL, 1.0 mL, 1.25 mL, 1.5 mL, up to 5 mL), and multiple-use kits may contain volumes within the midpoint and upper limits of the above ranges (e.g., 1.75 mL, 2.0 mL, 2.5 mL, 3.0 mL, 3.5 mL, 4.0 mL, 4.5 mL, and 5.0 mL). In certain embodiments, the kits disclosed herein may be configured to produce a multi-part processed human amniotic membrane composition in a volume of 1 cm. 2 : 1ml~2cm 2The multi-part processed human amniotic membrane composition comprises a micronized human amniotic membrane composition and an aqueous human amniotic fluid filtrate mixed in a ratio of 1:1 ml. In certain embodiments, the multi-part processed human amniotic membrane composition comprises a cryopreservation agent, the concentration of the cryopreservation agent being 1-10 wt% of the total concentration of the multi-part processed human amniotic membrane composition. For example, in certain embodiments, the cryopreservation agent may be dimethyl sulfoxide (DMSO) included in the multi-part processed human amniotic membrane composition at a total concentration of 1-10 wt% of the multi-part processed human amniotic membrane composition. Alternatively, the cryopreservation agent may be a DMSO-free cryopreservation agent (e.g., polyampholate, P24, etc.) at a total concentration of 1-10 wt% of the multi-part processed human amniotic membrane composition. Methods of Use of Multi-Part Processed Amniotic Membrane Compositions

[0115] Also disclosed herein are methods for treating Peyronie's disease in a subject in need of treatment. These methods include (a) preparing a multi-part processed human amniotic membrane composition configured to treat Peyronie's disease; and (b) injecting an effective amount of the multi-part processed human amniotic membrane composition configured to treat Peyronie's disease into the corpus cavernosum of a subject in need of treatment, thereby treating Peyronie's disease, relieving symptoms associated with Peyronie's disease, reducing plaque size associated with Peyronie's disease in a subject in need of treatment, and / or improving and / or restoring penile function. In certain embodiments, steps (a) and (b) disclosed immediately above are repeated at predetermined time periods until a desired result is obtained (i.e., relieving symptoms associated with Peyronie's disease, reducing plaque size associated with Peyronie's disease in a subject in need of treatment, and / or improving and / or restoring penile function).

[0116] In certain embodiments, the method comprises administering / injecting a multi-part processed human amniotic membrane composition configured to treat Peyronie's disease to a subject by internal injection, the multi-part processed human amniotic membrane composition comprising 1.2×10 6 pg / mL ~ 1.1 × 10 8 pg / mL and / or 1.3 × 10 6 pg / mL~5.5×107 pg / mL HA, 2.5 x 10 6 pg / mL~9×10 6 pg / mL and / or 2.75 × 10 6 pg / mL~5×10 6 pg / mL IGFBP-1, 5 × 10 7 pg / mL ~ 8.0 × 10 7 pg / mL and / or 5.3 × 10 7 pg / mL ~ 7.5 × 10 7 pg / mL of sGAG, particle diameter of 50-200 nm, and 1.0 × 10 9 pg / mL~2.0×10 9 pg / mL and / or 1.2 × 10 9 pg / mL ~ 1.7 × 10 9 Exosomes at a concentration of pg / mL, 2 × 10 4 pg / mL~1.5×10 5 pg / mL and / or 2.1×10 4 pg / mL~7×10 4 pg / mL IL-1ra, 900pg / mL to 3000pg / mL and / or 1000pg / mL to 1500pg / mL HGF, 1.25×10 4 ~2.25×10 4 pg / mL and / or 1.3 × 10 4 ~2.15×10 4 In certain embodiments, the method comprises administering / injecting a multi-part processed human amniotic membrane composition configured to treat Peyronie's disease to a subject by intracorporeal injection, the multi-part processed human amniotic membrane composition comprising 1.2×10 6 pg / mL ~ 1.1 × 10 8 pg / mL and / or 1.3 × 10 6 pg / mL~5.5×10 7 pg / mL HA and 2.5 × 10 6 pg / mL~9×10 6 pg / mL and / or 2.75 × 10 6 pg / mL~5×10 6 pg / mL IGFBP-1 and 5 × 107 pg / mL ~ 8.0 × 10 7 pg / mL and / or 5.3 × 10 7 pg / mL ~ 7.5 × 10 7 pg / mL of sGAG and a particle diameter of 50-200 nm, with a concentration of 1.0 × 10 9 pg / mL~2.0×10 9 pg / mL and / or 1.2 × 10 9 pg / mL ~ 1.7 × 10 9 pg / mL concentration of exosomes and 2 × 10 4 pg / mL~1.5×10 5 pg / mL and / or 2.1×10 4 pg / mL~7×10 4 pg / mL IL-1ra, 900pg / mL to 3000pg / mL and / or 1000pg / mL to 1500pg / mL HGF, and 1.25 × 10 4 ~2.25×10 4 pg / mL and / or 1.3 × 10 4 ~2.15×10 4 It contains pg / mL of transthyretin and the total volume is 250 μL to 5 mL.

[0117] The foregoing description provides embodiments of the present invention by way of example only. It is envisioned that other embodiments may perform a similar function and / or achieve similar results. All such equivalent embodiments and examples are intended to be included within the scope of the present invention and are covered by the appended claims.

Claims

1. 1. A multi-part processed human amniotic membrane composition adapted to treat Peyronie's disease by internal injection of an effective amount into a subject in need of treatment, comprising: (a) a micronized human amniotic membrane composition; (b) an aqueous human amniotic fluid filtrate configured to resuspend the micronized human amniotic membrane composition; and Equipped with The multi-part processed human amniotic membrane composition is a multi-part processed human amniotic membrane composition that was not treated with exogenous enzymes during its manufacture and does not contain added exogenous enzymes.

2. 2. The multi-part processed human amniotic membrane composition configured to treat Peyronie's disease by internal injection as described in claim 1, wherein the micronized human amniotic membrane composition is dried or pulverized micronized human amniotic membrane tissue.

3. 3. The multi-part processed human amniotic membrane composition adapted to treat Peyronie's Disease by internal injection according to claim 1 or 2, wherein the micronized human amniotic membrane composition has a particle diameter size of greater than 1 μm to less than 300 μm.

4. 4. The multi-part processed human amniotic membrane composition adapted to treat Peyronie's Disease by internal injection according to any one of claims 1 to 3, wherein the micronized human amniotic membrane composition has a particle diameter size of greater than 1 μm to 50 μm, and the particles are polydisperse.

5. 5. The multi-part processed human amniotic membrane composition adapted to treat Peyronie's Disease by internal injection according to any one of claims 1 to 4, wherein the micronized human amniotic membrane composition has a particle diameter size of greater than 1 μm to 25 μm, and the particles are polydisperse.

6. 6. The multi-part processed human amniotic membrane composition configured to treat Peyronie's Disease by internal injection according to any one of claims 1 to 5, wherein the micronized human amniotic membrane composition comprises hyaluronic acid (HA) and / or hyaluronan, fibronectin, insulin growth factor binding protein-1 (IGFBP-1), sulfated glycosaminoglycans (sGAGs), exosomes, interleukin-1 receptor antagonist (IL-1ra), hepatocyte growth factor (HGF), transthyretin, or any combination thereof.

7. The micronized human amniotic membrane composition comprises: dried micronized human amniotic membrane tissue having a particle diameter size of greater than 1 μm to less than 300 μm; Hyaluronic acid (HA) and / or hyaluronan, fibronectin, insulin growth factor binding protein-1 (IGFBP-1), sulfated glycosaminoglycans (sGAGs), exosomes, interleukin-1 receptor antagonist (IL-1ra), hepatocyte growth factor (HGF), transthyretin, or any combination thereof; A multi-part processed human amniotic membrane composition adapted for treating Peyronie's disease by internal injection according to any one of claims 1 to 6.

8. 8. The multi-part processed human amniotic membrane composition configured to treat Peyronie's Disease by internal injection according to any one of claims 1 to 7, wherein the aqueous human amniotic fluid filtrate comprises hyaluronic acid (HA) and / or hyaluronan, fibronectin, insulin growth factor binding protein-1 (IGFBP-1), sulfated glycosaminoglycans (sGAGs), exosomes, interleukin-1 receptor antagonist (IL-1ra), hepatocyte growth factor (HGF), transthyretin, or combinations thereof.

9. 9. The multi-part processed human amniotic membrane composition adapted to treat Peyronie's Disease by internal injection according to any one of claims 1 to 8, wherein the aqueous human amniotic fluid filtrate further comprises an isotonic solution.

10. 10. The multi-part processed human amniotic membrane composition adapted to treat Peyronie's Disease by internal injection according to any one of claims 1 to 9, wherein the isotonic solution is phosphate buffered saline.

11. 11. A multi-part processed human amniotic membrane composition adapted to treat Peyronie's Disease by internal injection according to any one of claims 1 to 10, wherein the aqueous human amniotic fluid filtrate comprises particles less than 70 μm in diameter.

12. The composition may be prepared by mixing the micronized human amniotic membrane composition with the aqueous human amniotic fluid filtrate at a concentration of 1.2×10 ６ ~1.1 x 10 ８ HA at a concentration of pg / mL, 2.5 x 10 ６ ~9×10 ６ pg / mL IGFBP-1, 5 x 10 ７ ~8.0 x 10 ７ pg / mL sGAG, with a particle diameter of 50-200 nm and 1.0 x 10 ９ ~2.0 x 10 ９ Exosomes at a concentration of 2 × 10 ４ ~1.5 x 10 ５ pg / mL IL-1ra, 900-3000 pg / mL HGF, 1.25 x 10 ４ ~2.25 x 10 ４ 12. A multi-part processed human amniotic membrane composition adapted to treat Peyronie's disease by internal injection according to any one of claims 1 to 11, comprising pg / mL transthyretin, ...

13. The composition may be prepared by mixing the micronized human amniotic membrane composition with the aqueous human amniotic fluid filtrate at a concentration of 1.2×10 ６ ~1.1 x 10 ８ pg / mL concentration of HA and 2.5 × 10 ６ ~9×10 ６ pg / mL IGFBP-1 and 5 × 10 ７ ~8.0 x 10 ７ pg / mL sGAG and a particle diameter of 50-200 nm and 1.0×10 ９ ~2.0 x 10 ９ and 2 × 10 ４ ~1.5 x 10 ５ pg / mL IL-1ra, 900-3000 pg / mL HGF, and 1.25 x 10 ４ ~2.25 x 10 ４ 13. A multi-part processed human amniotic membrane composition adapted to treat Peyronie's disease by internal injection according to any one of claims 1 to 12, comprising:

14. The micronized human amniotic membrane composition and the aqueous human amniotic fluid filtrate are mixed together in a volume of 1 cm ２ : 1ml to 2cm ２ 14. A multi-part processed human amniotic membrane composition adapted to treat Peyronie's disease by internal injection according to any one of claims 1 to 13, adapted to be mixed in a ratio of 1:1 ml.

15. 15. The multi-part processed human amniotic membrane composition adapted to treat Peyronie's Disease by internal injection according to any one of claims 1 to 14, wherein both the micronized human amniotic membrane composition and the aqueous human amniotic fluid filtrate are sterile.

16. 16. The multi-part processed human amniotic membrane composition configured to treat Peyronie's Disease by internal injection as described in claim 15, wherein both the micronized human amniotic membrane composition and the aqueous human amniotic fluid filtrate are non-immunogenic.

17. 17. The multi-part processed human amniotic membrane composition configured to treat Peyronie's Disease by internal injection of claim 16, wherein the multi-part processed human amniotic membrane composition reduces the size of Peyronie's Disease plaques.

18. 18. A multi-part processed human amniotic membrane composition adapted to treat Peyronie's Disease by internal injection according to any of claims 1 to 17, wherein in said multi-part processed human amniotic membrane composition transthyretin is present in an effective amount for treating Peyronie's Disease, for reducing symptoms associated with Peyronie's Disease, and / or for reducing the size of Peyronie's Disease plaques.

19. 20. The multi-part processed human amniotic membrane composition adapted to treat Peyronie's disease by internal injection according to claim 18, wherein in said multi-part processed human amniotic membrane composition, transthyretin is present at a concentration of 10-50 pg / mL.

20. 1. A kit comprising a multi-part processed human amniotic membrane composition adapted to treat Peyronie's disease by internal injection of an effective amount into a subject in need of treatment, a sterile container storing said multi-part processed human amniotic membrane composition adapted to treat Peyronie's disease by internal injection; 1. A kit comprising: a multi-part processed human amniotic membrane composition adapted to treat Peyronie's disease by internal injection, the kit comprising a micronized human amniotic membrane composition suspended in aqueous human amniotic fluid filtrate, and no added exogenous enzymes.

21. 21. The kit of claim 20, wherein the multi-part processed human amniotic membrane composition configured to treat Peyronie's Disease by internal injection does not contain collagenase.

22. 22. The kit of claim 20 or 21, wherein the micronized human amniotic membrane composition is dried or comminuted micronized human amniotic membrane tissue, preferably not subjected to exogenous enzymatic digestion.

23. The kit of any one of claims 20 to 22, wherein the micronized human amniotic membrane composition has a particle diameter of greater than 1 μm and less than 300 μm.

24. The kit of any one of claims 20-23, wherein the micronized human amniotic membrane composition has a particle diameter of greater than 1 μm to 50 μm, and the particles are polydisperse.

25. The kit of any one of claims 20-24, wherein the micronized human amniotic membrane composition has a particle diameter of greater than 1 μm to 25 μm, and the particles are polydisperse.

26. The kit of any one of claims 20 to 25, wherein the micronized human amniotic membrane composition comprises hyaluronic acid (HA) and / or hyaluronan, fibronectin, insulin growth factor binding protein-1 (IGFBP-1), sulfated glycosaminoglycan (sGAG), exosomes, interleukin-1 receptor antagonist (IL-1ra), hepatocyte growth factor (HGF), transthyretin, or any combination thereof.

27. The kit of any one of claims 20 to 26, wherein the micronized human amniotic membrane composition comprises hyaluronic acid (HA) and / or hyaluronan, fibronectin, insulin growth factor binding protein-1 (IGFBP-1), sulfated glycosaminoglycan (sGAG), exosomes, interleukin-1 receptor antagonist (IL-1ra), hepatocyte growth factor (HGF), and transthyretin.

28. The micronized amniotic membrane composition comprises: dried micronized human amniotic tissue having a particle diameter of greater than 1 μm and less than 300 μm; Hyaluronic acid (HA) and / or hyaluronan, fibronectin, insulin growth factor binding protein-1 (IGFBP-1), sulfated glycosaminoglycans (sGAGs), exosomes, interleukin-1 receptor antagonist (IL-1ra), hepatocyte growth factor (HGF), transthyretin, or any combination thereof; The kit according to any one of claims 20 to 27.

29. 29. The kit of any one of claims 20 to 28, wherein the aqueous human amniotic fluid filtrate comprises hyaluronic acid (HA) and / or hyaluronan, fibronectin, insulin growth factor binding protein-1 (IGFBP-1), sulfated glycosaminoglycans (sGAGs), exosomes, interleukin-1 receptor antagonist (IL-1ra), hepatocyte growth factor (HGF), transthyretin, or a combination thereof.

30. The kit of any one of claims 20 to 29, wherein the aqueous human amniotic fluid filtrate further comprises an isotonic solution.

31. The kit according to any one of claims 20 to 30, wherein the isotonic solution is phosphate buffered saline.

32. The kit of any one of claims 20 to 31, wherein the aqueous human amniotic fluid filtrate contains particles less than 100 μm in diameter.

33. The kit of any one of claims 20 to 32, wherein the micronized human amniotic membrane composition and the aqueous human amniotic fluid filtrate are mixed in a ratio of 2:1 to 1:

2.

34. The multi-part processed human amniotic membrane composition configured for treating Peyronie's disease by intracellular injection contains 1.2×10 ６ ~1.1 x 10 ８ HA at a concentration of pg / mL, 2.5 x 10 ６ ~9×10 ６ pg / mL IGFBP-1, 5 x 10 ７ pg / mL ~ 8.0×10 ７ pg / mL sGAG, with a particle diameter of 50-200 nm and 1.0 x 10 ９ ~2.0 x 10 ９ Exosomes at a concentration of 2 × 10 ４ ~1.5 x 10 ５ pg / mL IL-1ra, 900-3000 pg / mL HGF, 1.25 x 10 ４ ~2.25 x 10 ４ 34. The kit of any one of claims 20 to 33, comprising pg / mL transthyretin, pg / mL transthyretin, pg / mL transthyretin, or any combination thereof.

35. The multi-part processed human amniotic membrane composition configured for treating Peyronie's disease by intracellular injection contains 1.2×10 ６ ~1.1 x 10 ８ pg / mL concentration of HA and 2.5 × 10 ６ ~9×10 ６ pg / mL IGFBP-1 and 5 × 10 ７ pg / mL ~ 8.0×10 ７ pg / mL sGAG and a particle diameter of 50-200 nm and 1.0×10 ９ ~2.0 x 10 ９ and 2 × 10 ４ ~1.5 x 10 ５ pg / mL IL-1ra, 900-3000 pg / mL HGF, and 1.25 x 10 ４ ~2.25 x 10 ４ The kit of any one of claims 20 to 34, comprising pg / mL transthyretin.

36. 36. The kit of any one of claims 20-35, wherein the multi-part processed human amniotic membrane composition configured to treat Peyronie's Disease is preservative and / or sterile.

37. 37. The kit of any one of claims 20-36, wherein the multi-part processed human amniotic membrane composition adapted to treat Peyronie's Disease is non-immunogenic.

38. 38. The kit of any one of claims 20-37, wherein the multi-part processed human amniotic membrane composition configured to treat Peyronie's Disease, for alleviating symptoms associated with Peyronie's Disease and / or for reducing the size of Peyronie's Disease plaques.

39. 39. The kit of any one of claims 20-38, wherein transthyretin is present in the multi-part processed human amniotic membrane composition in an effective amount for treating Peyronie's Disease, reducing symptoms associated with Peyronie's Disease, and / or reducing the size of Peyronie's Disease plaques.

40. The kit of any one of claims 20 to 39, wherein transthyretin is present in the multi-part processed human amniotic membrane composition at a concentration of 10 to 50 pg / mL.

41. 1. A method of treating Peyronie's disease in a subject in need thereof, comprising: (a) providing the multi-part processed human amniotic membrane composition adapted to treat Peyronie's Disease according to claim 1 or claim 18; (b) injecting an effective amount of the multi-part processed human amniotic membrane composition configured to treat Peyronie's Disease into the corpus cavernosum of the subject in need of treatment to treat Peyronie's Disease in the subject in need of treatment, reduce symptoms associated with Peyronie's Disease, and / or reduce plaque size associated with Peyronie's Disease; A method comprising:

42. The multi-part processed human amniotic membrane composition configured to treat Peyronie's disease by intracorporeal injection contains 1.2×10 ６ ~1.1 x 10 ８ HA at a concentration of pg / mL, 2.5 x 10 ６ ~9×10 ６ pg / mL IGFBP-1, 5 x 10 ７ pg / mL ~ 8.0×10 ７ pg / mL sGAG, with a particle diameter of 50-200 nm and 1.0 x 10 ９ ~2.0 x 10 ９ Exosomes at a concentration of 2 × 10 ４ ~1.5 x 10 ５ pg / mL IL-1ra, 900-3000 pg / mL HGF, 1.25 x 10 ４ ~2.25 x 10 ４ 42. The method of claim 41 , wherein the total volume is between 250 μL and 5 mL and the total volume is between 250 μL and 5 mL.

43. The multi-part processed human amniotic membrane composition configured to treat Peyronie's disease by intracorporeal injection contains 1.2×10 ６ ~1.1 x 10 ８ pg / mL concentration of HA and 2.5 × 10 ６ ~9×10 ６ pg / mL IGFBP-1 and 5 × 10 ７ pg / mL ~ 8.0×10 ７ pg / mL sGAG and a particle diameter of 50-200 nm and 1.0×10 ９ ~2.0 x 10 ９ and 2 × 10 ４ ~1.5 x 10 ５ pg / mL IL-1ra, 900-3000 pg / mL HGF, and 1.25 x 10 ４ ~2.25 x 10 ４ 42. The method of claim 41, comprising: pg / mL transthyretin in a total volume of 250 μL to 5 mL.

44. 1. A method of producing a multi-part processed human amniotic membrane composition adapted to treat Peyronie's disease by internal injection into a subject in need of treatment, comprising: (a) providing a fresh human amniotic membrane; (b) separating the human amniotic membrane from other human placental components; and (c) rinsing the separated human amniotic membrane of step (b) with alcohol; (d) washing the separated human amniotic membrane of step (c) with an isotonic solution; (e) drying the separated human amniotic membrane of step (d) at ambient temperature in a biosafety cabinet with a circulating fan for 2-6 hours or at a temperature of 30° C.-40° C. for 15 minutes-1 hour in a dehydrator to produce a dried human amniotic membrane; (f) milling the dried human amniotic membrane to produce a micronized human amniotic membrane composition having a particle diameter of 1-500 microns; (a') providing fresh human amniotic fluid; (b') filtering the fresh human amniotic fluid through a 200 micron filter to produce a first human amniotic fluid filtrate; (c') filtering the first human amniotic fluid filtrate through a 100 micron filter to produce a second human amniotic fluid filtrate; (d') filtering the second human amniotic fluid filtrate through a 70 micron filter to produce a third human amniotic fluid filtrate; (f') optionally diluting the third human amniotic fluid filtrate with a predetermined amount of a balanced salt solution to produce a diluted third human amniotic fluid filtrate; (i) mixing the micronized human amniotic membrane composition with either the third human amniotic fluid filtrate or the diluted third human amniotic fluid filtrate to produce the multi-part processed human amniotic membrane composition configured for treating Peyronie's disease by intracorporeal injection; Including, A method wherein none of the steps involves the introduction of an exogenous enzyme.

45. 45. The method of claim 44, wherein fresh human amniotic membrane is obtained from a subject and steps (a)-(e) are then performed within 48-72 hours after birth and / or Caesarean section.

46. 46. ​​The method of claim 44 or 45, further comprising removing a thrombus present within the human amniotic membrane during steps (a) to (c).

47. 47. The method of any one of claims 44 to 46, wherein step (d) is repeated 1 to 5 times by discarding the used isotonic solution, preparing a new isotonic solution, and washing the human amniotic membrane again with the new isotonic solution.

48. 48. The method of any one of claims 44 to 47, wherein the isotonic solution is phosphate buffered saline provided in a volume of from 300 mL to 1000 mL per wash step (d).

49. 49. The method of any one of claims 44-48, wherein in step (f), the dried human amniotic membrane is milled using a milling tool configured to mill and / or chop the dried human amniotic membrane at 40-200 revolutions per minute (RPM) until the dried human amniotic membrane is comminuted to form the micronized human amniotic membrane composition having a particle diameter of 1-500 microns.

50. The multi-part processed human amniotic membrane composition configured to treat Peyronie's disease by intracorporeal injection contains 1.2×10 ６ ~1.1 x 10 ８ HA at a concentration of pg / mL, 2.5 x 10 ６ ~9×10 ６ pg / mL IGFBP-1, 5 x 10 ７ pg / mL ~ 8.0×10 ７ pg / mL sGAG, with a particle diameter of 50-200 nm and 1.0 x 10 ９ ~2.0 x 10 ９ Exosomes at a concentration of 2 × 10 ４ ~1.5 x 10 ５ pg / mL IL-1ra, 900-3000 pg / mL HGF, 1.25 x 10 ４ ~2.25 x 10 ４ 50. The method of any one of claims 44-49, comprising pg / mL transthyretin, pg / mL transthyretin, or any combination thereof.

51. The multi-part processed human amniotic membrane composition configured to treat Peyronie's disease by intracorporeal injection contains 1.2×10 ６ ~1.1 x 10 ８ pg / mL concentration of HA and 2.5 × 10 ６ ~9×10 ６ pg / mL IGFBP-1 and 5 × 10 ７ pg / mL ~ 8.0×10 ７ pg / mL sGAG and a particle diameter of 50-200 nm and 1.0×10 ９ ~2.0 x 10 ９ and 2 × 10 ４ ~1.5 x 10 ５ pg / mL IL-1ra, 900-3000 pg / mL HGF, and 1.25 x 10 ４ ~2.25 x 10 ４ 51. The method of any one of claims 44 to 50, comprising pg / mL transthyretin.

52. 52. The method of any one of claims 44-51, wherein transthyretin is present in the multi-part processed human amniotic membrane composition in an amount effective to treat Peyronie's Disease, to reduce symptoms associated with Peyronie's Disease, and / or to reduce the size of Peyronie's Disease plaques.

53. 53. The method of any one of claims 44-52, wherein transthyretin is present in the multi-part processed human amniotic membrane composition at a concentration of 10-50 pg / mL.

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