Pharmaceutical composition for treating degenerative ocular diseases and use thereof

Abstract

A pharmaceutical composition containing a stem cell secretion, and a pharmaceutically acceptable excipient thereof. The protein in the stem cell secretion comprises one, two or three proteins selected from a group consisting of a pigment epithelium-derived factor (PEDF), a tissue inhibitor of metalloproteinase 1 (TIMP-1), a secreted protein acidic and rich in cysteine (SPARC), an α2-HS glycoprotein (AHSG), a periostin (POSTN), a decorin (DCN), the retinoic acid receptor responder 2 (RARRES2), the glia-derived nexin (GDN), the Sparc / osteonectin, cwcv and kazal like domains proteoglycan 1 (SPOCK1), and the follistatin-like 1 (FSTL1). The pharmaceutical composition is used for treating degenerative ocular diseases including retinal degeneration, retinitis pigmentosa, glaucoma, macular degeneration, etc.

Description

Pharmaceutical compositions for treating degenerative ocular diseases and uses thereof TECHNICAL FIELD

[0001] The present disclosure relates to pharmaceutical compositions comprising stem cell secretomes, and uses and methods of using the same for treating degenerative ocular diseases. BACKGROUND

[0002] The retina is a laminated tissue composed of six distinct types of neural cells stacked in series, each responsible for processing visual signals and forming synaptic relays to transduce visual light into the visual cortex of the brain to form vision. Any ocular injury or ocular pathology can cause the death of retinal neurons, especially photoreceptors and retinal pigment epithelial cells, which are responsible for maintaining and regulating different types of neurons. Since these neural cells cannot be regenerated after loss, any damage to the neural cells caused by any cause can lead to irreversible visual impairment, even blindness.

[0003] For example, age-related macular degeneration (AMD) is a very common disease, which occurs when the macula, the central part of the retina with a high density of photoreceptors, responsible for the most important visual function, is degenerated, resulting in a significant reduction in visual acuity and thus a dramatic impact on life. In addition, retinitis pigmentosa (RP) is a common congenital hereditary retinal degeneration that gradually reduces due to the spontaneous death of photoreceptors, leading to night blindness and reduced visual field, eventually causing severe visual impairment, even vision loss.

[0004] The treatment of retinal degeneration-related disorders in the clinic is quite limited, and most of them are incurable diseases. For example, the only approved treatment for retinitis pigmentosa is Luxturna, which targets the RPE65 gene. However, there are currently 65 known genetic mutations that cause retinitis pigmentosa, but the patient population that Luxturna can treat is only about 2-3% of all retinitis pigmentosa patients. Therefore, most patients with retinitis pigmentosa still have no effective treatment drugs to date.

[0005] In recent years, cell therapy has progressed rapidly. Although many studies have confirmed that mesenchymal stem cell treatment is safe and effective for retinal degenerative diseases, recent studies have also found that mesenchymal stem cell treatment still has potential problems to overcome, including the heterogeneity of mesenchymal stem cells due to cell source, separation method and culture method, which may affect the characteristics of mesenchymal stem cells and thus produce differences in treatment effect. In addition, the low survival rate of mesenchymal stem cells in the body may result in mesenchymal stem cells being unable to provide long-term protection and treatment effect. Furthermore, the inflammatory microenvironment of mesenchymal stem cells will differentiate into Th17 cells of the pro-inflammatory type, thus producing a more severe inflammatory response. In addition, the injection of mesenchymal stem cells into the eye may affect the survival rate of the cells and cause side effects.

[0006] It was also found that the injection of human adipose stem cells into the eyes of rats would cause serious adverse reactions, such as growth retardation and death, and it was speculated that these serious adverse reactions might be related to rejection between the graft and the host. In addition, according to the autopsy report, it was found that the brain, lungs and muscles of the dead rats showed extensive flushing, and a large number of immune cell infiltrations were found in the muscles and lungs. In addition, the report also pointed out that there were problems such as atrophy of the epidermis, hair follicles and accessory glands, and the results of staining of the sampling sections of the periocular tissues of the rats showed that a large number of CD8+ T cell signals were observed in the periocular tissues, indicating that the rats produced an immune rejection reaction after injection of human adipose stem cells. In addition, after collecting blood samples from rats injected with human adipose stem cells in the eye and analyzing them, it was found that a large number of IL-18 and CXCL10 were expressed, indicating that the injection of human adipose stem cells into the eye indeed caused rejection of the host. Therefore, the injection of mesenchymal stem cells into the eye for the treatment of retinitis pigmentosa not only fails to improve the symptoms of eye disease, but also may cause serious health hazards.

[0007] Therefore, there is still a need for a safe and effective treatment for degenerative eye diseases that provides treatment for degenerative eye diseases with low side effects. SUMMARY

[0008] The present disclosure provides a pharmaceutical composition comprising stem cell secretome and a pharmaceutically acceptable excipient, wherein the stem cell secretome comprises at least one protein selected from the group consisting of pigment epithelium-derived factor (PEDF), tissue inhibitor of metalloproteinase 1 (TIMP-1), secreted protein acidic and rich in cysteine (SPARC), alpha2-HS glycoprotein (AHSG), periostin (POSTN), decorin (DCN), retinoic acid receptor response 2 (RARRES2), glia-derived nexin (GDN), bone morphogenetic protein 1 (BMP1), and follistatin-related protein 1 (FSTL1).

[0009] In some embodiments of the present disclosure, the stem cell secretome contained in the pharmaceutical composition comprises one, two or three proteins selected from the group consisting of pigment epithelium-derived factor (PEDF), tissue inhibitor of metalloproteinase 1 (TIMP-1), secreted protein acidic and rich in cysteine (SPARC), alpha2-HS glycoprotein (AHSG), periostin (POSTN), decorin (DCN), retinoic acid receptor response 2 (RARRES2), glia-derived nexin (GDN), bone morphogenetic protein 1 (BMP1), and follistatin-related protein 1 (FSTL1).

[0010] In some embodiments of the present disclosure, the stem cell secretome contained in the pharmaceutical composition comprises at least two proteins selected from the group consisting of pigment epithelium-derived factor (PEDF), tissue inhibitor of metalloproteinase 1 (TIMP-1), secreted protein acidic and rich in cysteine (SPARC), alpha2-HS glycoprotein (AHSG), periostin (POSTN), decorin (DCN), retinoic acid receptor response 2 (RARRES2), glia-derived nexin (GDN), bone morphogenetic protein 1 (BMP1), and follistatin-related protein 1 (FSTL1). In some embodiments of the present disclosure, the stem cell secretome comprises secreted protein acidic and rich in cysteine (SPARC). In some embodiments of the present disclosure, the stem cell secretome comprises pigment epithelium-derived factor (PEDF), periostin (POSTN), and secreted protein acidic and rich in cysteine (SPARC).

[0011] In some embodiments of the disclosure, the stem cell secretome comprised in the pharmaceutical composition is a mesenchymal stem cell secretome. In some embodiments of the disclosure, the stem cell secretome comprised in the pharmaceutical composition is an adipose-derived mesenchymal stem cell secretome. In some embodiments of the disclosure, the stem cell secretome is obtained from stem cells cultured in vitro. In some embodiments of the disclosure, the stem cell secretome is present in a conditioned medium of stem cells cultured in vitro. In some embodiments of the disclosure, the conditioned medium of stem cells cultured in vitro comprises the stem cell secretome.

[0012] In at least one embodiment of the disclosure, the pharmaceutical composition provided by the disclosure is a cell-free composition. For example, in some embodiments of the disclosure, the cell-free composition does not contain cells, or cells are removed after treatment.

[0013] In at least one embodiment, the disclosure further provides a use of a pharmaceutical composition for the preparation of a medicament for treating degenerative eye diseases, wherein the pharmaceutical composition comprises a stem cell secretome and a pharmaceutically acceptable excipient thereof, wherein the stem cell secretome comprises at least one protein from the group consisting of pigment epithelium-derived factor (PEDF), tissue inhibitor of metalloproteinase 1 (TIMP-1), cysteine-rich acidic secreted protein (SPARC), alpha 2-HS glycoprotein (AHSG), periostin (POSTN), decorin (DCN), retinoic acid receptor response protein 2 (RARRES2), glia-derived nexin (GDN), bone morphogenetic protein 1 (BMP1), and follistatin-like protein 1 (FSTL1).

[0014] In at least one embodiment, the method provided by the disclosure comprises intravitreal administration of the pharmaceutical composition to a subject. In some embodiments of the disclosure, the pharmaceutical composition is injected into the vitreous for administration to a subject in need thereof. In some embodiments, the use provided by the disclosure delays retinal degeneration in a subject. In at least one embodiment, the retinal degeneration in the subject is delayed for at least one month. In at least one embodiment, the retinal degeneration in the subject is delayed for at least two months.

[0015] In some embodiments of the disclosure, the use provided by the disclosure protects retinal function in a subject. In at least one embodiment, the protection of retinal function in the subject lasts for at least one month. In at least one embodiment, the protection of retinal function in the subject lasts for at least two months.

[0016] In at least one embodiment, the degenerative eye disease of the present disclosure is retinal degeneration, retinitis pigmentosa, glaucoma, or macular degeneration. In some embodiments of the present disclosure, the degenerative eye disease is retinitis pigmentosa, dry macular degeneration, or age-related macular degeneration.

[0017] In at least one embodiment, the subject of the present disclosure is a mammal, such as a human.

[0018] In at least one embodiment, the present disclosure further provides a method of treating a degenerative eye disease, comprising administering to a subject in need thereof a pharmaceutical composition comprising an effective amount of stem cell secretome, wherein the stem cell secretome comprises at least one protein from the group consisting of pigment epithelium-derived factor (PEDF), tissue inhibitor of metalloproteinase 1 (TIMP-1), cysteine-rich acidic secreted protein (SPARC), alpha 2-HS glycoprotein (AHSG), periostin (POSTN), decorin (DCN), retinoic acid receptor response protein 2 (RARRES2), glia-derived nexin (GDN), bone morphogenetic protein 1 (BMP1), and follistatin-related protein 1 (FSTL1). In some embodiments of the present disclosure, the degenerative eye disease is retinal degeneration, retinitis pigmentosa, glaucoma, or macular degeneration. In some embodiments of the present disclosure, the degenerative eye disease is retinitis pigmentosa, dry macular degeneration, or age-related macular degeneration.

[0019] It is to be understood that the foregoing general description and the following detailed description are merely intended to introduce the reader to the concepts described in further detail below, and are not intended to be limiting or key features of the present disclosure, the scope of which is defined by the appended claims. Furthermore, the objectives of the present disclosure are not limited to embodiments that solve any or all of the problems described in the background section or elsewhere in this specification. The present disclosure includes combinations of any of the features disclosed herein, whether explicitly mentioned or not. Furthermore, if a combination of two or more features is mentioned, for example, in the same paragraph, the features can be requested separately, without expanding the scope of the present disclosure. Features of the different embodiments described below can also be combined in any claim. BRIEF DESCRIPTION OF DRAWINGS

[0020] The present disclosure can be implemented in a number of ways, illustrative embodiments of which are described below and which are illustrated in the accompanying drawings.

[0021] Figure 1 shows the results of retinal tomography analysis and the statistical results of retinal thickness (RT) of Wistar rats injected with conditioned medium containing stem cell secretions (Exofectin-5) and PBS, respectively, intravitreal injection (IVI) 28 days later.

[0022] Figure 2 shows the results of electroretinogram analysis of Wistar rats injected with conditioned medium containing stem cell secretions (Exofectin-5) and PBS, respectively, intravitreal injection (IVI) before injection, and 28 days after injection (D28).

[0023] Figure 3 shows the results of electroretinogram analysis of Royal College of Surgeon rats (RCS rats) injected with conditioned medium containing stem cell secretions (Exofectin-5) and PBS, respectively, intravitreal injection (IVI) 56 days later.

[0024] Figure 4 shows the results of retinal tomography analysis and the statistical results of retinal thickness (RT) of Royal College of Surgeon rats (RCS rats) injected with conditioned medium containing stem cell secretions (Exofectin-5) and PBS, respectively, intravitreal injection (IVI) 56 days later.

[0025] Figure 5 shows 52 proteins with stable high expression levels in conditioned medium containing stem cell secretions and their interactions.

[0026] Figure 6 shows the results of absolute concentration determination of 10 proteins with stable expression in different batches of conditioned medium by enzyme-linked immunosorbent assay (ELISA).

[0027] Figure 7 shows the results of cell survival rate determination by CCK-8 cell survival rate assay after treatment with PEDF or POSIN protein after high oxidative stress test with human retinal pigment epithelial cells ARPE-19.

[0028] Figure 8 shows the results of cell staining for mitochondrial superoxide and intracellular oxidation after staining with JC-1 after treatment with PEDF, POSIN or SPARC protein after cell aging and degeneration test with human retinal pigment epithelial cells ARPE-19. DETAILED DESCRIPTION

[0029] Unless otherwise defined in this specification, scientific and technical terms as used herein have the same meaning as understood and commonly used by those skilled in the art. Furthermore, unless contextually conflicting, singular nouns as used herein encompass plural forms of the same noun, and plural forms may encompass singular forms of the same noun. Specifically, in this specification and the claims, the singular forms “a” or “the” include plural forms unless the context clearly indicates otherwise.

[0030] As used herein, the term "at least one" in relation to a list of one or more elements should be understood to mean at least one element selected from any one or more elements in the element list, but does not necessarily include at least one of each element listed in the element list, and does not exclude any combination of elements in the element list. The definition also allows for the optional presence of elements other than those referred to in the element list by the term "at least one," whether related to or unrelated to the referred element. Thus, as a non-limiting example, "at least one of A and B" (or equivalently, "at least one of A or B," or equivalently, "at least one of A and / or B") in one embodiment may refer to at least one, optionally including more than one A, while B is absent (and optionally includes elements other than B); in another embodiment may refer to at least one, optionally including more than one B, while A is absent (and optionally includes elements other than A); and in yet another embodiment may refer to at least one, optionally including more than one A, and at least one, optionally including more than one B (and optionally including other elements). Furthermore, in this specification and the claims, the terms "at least one" and "one or more" have the same meaning, both representing a total of one, two, three or more.

[0031] The terms “at least” and similar nouns, such as “at least one” or “more”, used in this specification shall be understood to include (but not limited to) at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 300, 400, 500, 600, 700, 800, 900, 1,000, 2,000, 3,000, 4,000, 5,000 or more of the stated values, and also include any larger numbers or fractions thereof.

[0032] The terms “for example” and “that is” used in this specification are for illustrative purposes only and are not intended to be limiting, nor should they be construed as referring only to the items explicitly listed in this specification.

[0033] The terms "comprise", "have", "contain", or variants such as "comprising", "including", "containing", "including" should be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

[0034] The practice of the present disclosure employs, unless otherwise indicated, conventional techniques of molecular biology, cell biology, biochemistry and immunology within the skill of the art. Such techniques are explained fully in the literature, for example, "Molecular Cloning: A Laboratory Manual" Second Edition (Sambrook, et al., 1989), Cold Spring Harbor Press; "Oligonucleotide Synthesis" (M.J. Gait, 1984); "Methods in Molecular Biology", Humana Press; "Cell Biology: A Laboratory Notebook" (J.E. Cellis, Ed., 1998), Academic Press; "Animal Cell Culture" (R.I. Freshney, Ed., 1987); "Handbook of Experimental Immunology" (Weir, 1996); "Introduction to Cell and Tissue Culture" (J.P. Mather and P.E. Roberts, 1998); "Cell and Tissue Culture: Laboratory Procedures" (A. Doyle, J.B. Griffiths and D.G. Newell, Eds., 1993-8); "Methods in Enzymology" (Academic Press, Inc.); "Handbook of Experimental Immunology" (D.M. Weir and C.C. Blackwell, Eds.); "Gene Transfer Vectors for Mammalian Cells" (J.M. Miller and M.P. Calos, Eds., 1987); "Current Protocols in Molecular Biology" (F.M. Ausubel, et al., Eds., 1987); "PCR: The Polymerase Chain Reaction" (Mullis, et al., Eds., 1994); "Current Protocols in Immunology" (J.E. Coligan et al., Eds., 1991); "Short Protocols in Molecular Biology" (Wiley & Sons, 1999); "Immunobiology" (C. A. Janeway and P. Travers, 1997); "Antibodies" (P. Finch, 1997); "Antibodies: A Practical Approach" (D. Catty, Ed., IRL Press, 1988-1989); "Monoclonal Antibodies: A Practical Approach" (P. Shepherd and C. Dean, Eds., Oxford University Press, 2000); "Using Antibodies: A Laboratory Manual" (E. Harlow and D. Lane, Cold Spring Harbor Laboratory Press, 1999); and "The Antibodies" (M. Zanetti and J. D. Capra, Eds., Harwood Academic Publishers, 1995). Specific techniques used for particular embodiments will be described in the following paragraphs. To the extent not further explained, it is believed that one of ordinary skill in the art will employ the disclosure in light of the above description to its fullest extent. Accordingly, the following specific embodiments are to be regarded as illustrative only and are not limiting on the remainder of the disclosure in any way. All publications cited herein are incorporated by reference for the purposes of the subject matter and content disclosed therein.

[0035] The terms "about," "approximately," and "substantially" generally mean within 10%, 5%, 1%, or 0.5% of a given value or range. Alternatively, the terms "about," "approximately," and "substantially" mean within standard error of the mean for the particular technology or discipline, unless otherwise indicated. Unless otherwise indicated, all numerical ranges disclosed herein are to be understood as being modified in all instances by the term "about," unless otherwise indicated. Thus, any numerical value, range, or percentage disclosed herein should be understood as being approximate, unless otherwise indicated.

[0036] The phrase "administering" or "administered" means introducing a pharmaceutical preparation into a subject using any of a variety of methods and delivery systems known to the skilled artisan. Exemplary routes of administration of the pharmaceutical preparations disclosed herein include intra-arterial, intravenous, intramuscular, subcutaneous, intraperitoneal, intraspinal, intratumoral, or other parenteral routes of administration, such as by injection or infusion. As used herein, the phrase "parenteral administration" means modes of administration other than enteral and topical, and includes, without limitation, intravenous, intramuscular, intra-arterial, intrathecal, intralymphatic, intralesional, intratumoral, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraepidermal, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, sternal, intraocular, intravitreal, subconjunctival, and subretinal injections and infusions, as well as in vivo electroporation. In some embodiments, the pharmaceutical preparation is administered by enteral routes, such as oral administration. Other enteral routes include topical, dermal, or transmucosal routes of administration, such as intranasal, vaginal, rectal, sublingual, or topical. Administration can be, for example, one, multiple, and / or over one or more extended periods of time. In some embodiments, the cell therapy provided by the present disclosure is administered via an "infusion product" comprising the pharmaceutical preparation of the present disclosure.

[0037] In the present disclosure, the terms "individual" and "subject" are used interchangeably and mean an animal, such as a mammal, including a human. The term "subject" is intended to encompass both males and females, e.g., men and women, unless explicitly indicated to the contrary. Non-limiting examples of non-human animal subjects include: rodents, such as mice, rats, hamsters, and guinea pigs; rabbits; dogs; cats; sheep; pigs; goats; cows; horses; and non-human primates, such as apes and monkeys.

[0038] The phrases "reducing" and "reduction" are also used interchangeably herein and mean any change from a starting state. "Reducing" and "reduction" are relative terms requiring comparison between pre-measurement and post-measurement. Similarly, the term "increasing" indicates any change above an initial value. "Increasing," "higher," and "lower" are relative terms requiring comparison between pre-measurement and post-measurement and / or to a reference standard. In some embodiments, the reference value is obtained from a reference value of a general population, which can be a general patient population.

[0039] "Treating" a subject means performing any type of intervention or process on the subject, or administering a preparation to the subject, with the goal of reversing, alleviating, ameliorating, inhibiting, slowing, or stopping the onset, progression, development, severity, or recurrence of a symptom, complication, or condition, or a biochemical marker associated with a disease. In some embodiments, "treating" includes partial alleviation of a disorder. In other embodiments, "treating" includes complete alleviation of a disorder.

[0040] The term "pharmaceutical composition" of the present specification means a composition in a form suitable for administration to a subject in need of treatment, and which contains an active ingredient in an effective amount to achieve the intended biological activity without undue toxicity to the subject to which it is administered. In the present specification, "pharmaceutical composition" means a medicament generally used for the treatment or prevention of a disease. The pharmaceutical preparation of the present disclosure can be prepared in a manner known to those skilled in the art. Furthermore, the pharmaceutical composition of the present disclosure containing stem cell secretions can also be formulated in combination with other pharmaceutical ingredients as needed. For example, a sterile solution using water or other pharmaceutically acceptable liquids, or an injection agent in the form of a suspension agent for non-oral use, for example, a pharmaceutically acceptable carrier or medium, such as a suitable combination with sterilized water, physiological saline, vegetable oil, emulsifier, suspending agent, surfactant, stabilizer, flavoring agent, excipient, carrier, preservative, binding agent, etc., can be mixed in a generally recognized unit dosage form required for the practice of pharmacy, and thereby formulated, and the amount of the active ingredient in such a formulation can be set to an appropriate amount in the indicated range.

[0041] The term "effective amount" of the present specification is an amount sufficient to achieve a beneficial or desired result. For example, a therapeutic amount is an effective amount to achieve a desired therapeutic effect. The amount can be the same as or different from a prophylactically effective amount, which is an amount necessary to prevent the onset of a disease or symptoms of a disease. An effective amount can be administered once or more than once. The therapeutically effective amount of a therapeutic compound (i.e., effective dose) depends on the selected therapeutic compound. The composition can be administered once or more than once a day to once or more than once a week, including once every two days. Those skilled in the art will appreciate that certain factors can influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and / or age of the subject, and the presence of other diseases. Moreover, treatment of a subject with a therapeutically effective amount of a therapeutic compound disclosed herein can include a single treatment or a series of treatments.

[0042] The term "cell-free" of the present specification means a state in which there are no or almost no cells.

[0043] The term "conditioned medium" of the present specification is a medium in which a specific cell or cell population is cultured and then removed. When cells are cultured in a medium, they can secrete cytokines that provide nutritional support to other cells, such as hormones, interleukins, extracellular matrix, proteins, vesicles, antibodies, and granules, and the conditioned medium can be a medium containing cytokines.

[0044] The term "degenerative eye disease or condition" of the present specification is an inclusive term encompassing acute and chronic conditions, disorders, or diseases of the eye (including neural connections between the eye and the brain) involving cell damage, degeneration, or loss. Degenerative eye diseases or eye degenerative conditions can be age-related, or can result from injury or trauma, or can be associated with a particular disease or disorder. Acute eye degenerative conditions include, but are not limited to, conditions associated with cell death or damage affecting the eye, including conditions resulting from cerebrovascular insufficiency, focal or diffuse brain trauma, diffuse brain injury, infectious or inflammatory eye conditions, retinal tears or detachments, intraocular lesions (contusion penetration, compression, laceration), or other physical injuries (e.g., physical or chemical burns). Chronic degenerative eye diseases or eye degenerative conditions (including progressive conditions) include, but are not limited to, retinal diseases and other retinal / macular disorders such as retinitis pigmentosa (RP; also known as "pigmentary retinopathy"), age-related macular degeneration (AMD; also known as "age-related macular degeneration"), choroidal neovascular membrane (CNVM); retinal diseases such as diabetic retinopathy, occlusive retinal disease, sickle cell retinal disease, and hypertensive retinal disease; central retinal vein occlusion, carotid artery stenosis; optic nerve diseases such as glaucoma and related syndromes; diseases of the crystalline lens and the outer eye such as limbal stem cell deficiency (LSCD), also known as limbal epithelial cell deficiency (LECD), for example occurring in chemical or thermal injury, contact lens-induced keratopathy, ocular cicatricial pemphigoid, congenital aniridia or ectodermal dysplasia diseases, and multiple endocrine deficiency-related keratitis.

[0045] For the purpose of making the present disclosure more thorough and complete, the following describes embodiments of the present disclosure and specific examples thereof, but this is not the only form of implementing or using the embodiments of the present disclosure. The embodiments include features of the specific embodiments and methods for constructing and operating the specific embodiments and the order thereof, but other embodiments can also be used to achieve the same or equivalent functions and order of steps.

[0046] Example 1: Preparation of a pharmaceutical composition comprising stem cell secretions

[0047] This example provides an example of a method for culturing adipose-derived mesenchymal stem cells, and a method for obtaining stem cell secretions thereof.

[0048] First, the isolation and purification of adipose-derived stem cells are performed, which includes washing the fat, removing the lower blood water waste liquid after the fat is layered, and transferring the fat to a centrifuge tube for centrifugation. Then, the fat block is poured into a plate, cut into small pieces, and transferred to a centrifuge tube. After adding collagenase, the mixture is placed in a culture box and slowly rotated to mix evenly. The mixture is filtered into a centrifuge tube and centrifuged. After centrifugation, the cell precipitate is removed and washed. Then, the cell precipitate is dispersed in Dulbecco's phosphate-buffered saline (dPBS) and centrifuged again. The supernatant after centrifugation is subjected to sterile test and mycoplasma and endotoxin content test. The adipose-derived stem cells obtained after centrifugation are cultured in a conventional culture bottle with a culture solution containing 5 to 20% human platelet lysate (hPL) according to the cell number and condition.

[0049] After obtaining the adipose-derived mesenchymal stem cells, the cells are expanded and cultured in a conventional culture vessel (e.g., a T-Flask culture bottle or a Culture Dish culture plate) using cell culture technology. Then, a bioreactor is used for adipose-derived stem cell culture and collection of stem cell secretions, including: (1) removing the old culture solution in the original culture container; (2) washing the cells twice with dPBS; (3) adding an appropriate amount of trypsin and ethylenediaminetetraacetic acid (EDTA) solution to suspend all cells; (4) centrifuging the cell suspension at 300 to 500 x g for 3 to 10 minutes after neutralizing with an appropriate amount of culture solution; (5) removing the supernatant, retaining the cells at the bottom of the tube, and suspending the cells by washing with culture solution at a concentration of about 10 to 15 million cells per milliliter; (6) uniformly distributing the cell suspension on three-dimensional carriers in a bioreactor-specific bottle, in which at least 500 three-dimensional carriers are used; (7) placing the bioreactor-specific bottle in a 37°C, 5% CO2 incubator for at least 60 minutes, and then adding DMEM / F12 culture solution containing at least 10% fetal bovine serum to the bioreactor-specific bottle and placing it in the incubator for at least 2 days. In this example, the culture solution components used can include various culture solutions, such as Dulbecco's Modified Eagle Medium (DMEM), DMEM / F12, F12, StemPro, fetal bovine serum (FBS), and antibiotics (e.g., gentamicin or penicillin), similar to the conventional culture dish method, which will not be described here.

[0050] During the culturing period, the cells are washed and replaced with a buffer solution, such as dPBS or PBS, every 48 to 72 hours. Then, the bioreactor-specific bottle is connected to a serum bottle containing the culture solution, and the connected line is installed on a peristaltic pump. The cell culture solution is perfused at a constant rate and amount. After culturing the cells in serum-free culture solution for at least 48 hours, the culture solution containing the secretions of the adipose-derived stem cells can be collected.

[0051] Example 2: Safety evaluation of the pharmaceutical composition containing stem cell secretions

[0052] To confirm the safety of the pharmaceutical composition containing stem cell secretions according to the present disclosure, the conditioned medium containing stem cell secretions (Exofectin-5) and PBS as a control group were injected into the vitreous, subconjunctival, or subretinal position of rats, respectively. After 28 days from the completion of the injection, the retinal structure and the change in the retinal thickness of Wistar rats were examined using optical coherence tomography (OCT). The results are shown in FIG. 1. Injection of 5 μL of the conditioned medium containing stem cell secretions into the vitreous did not cause changes in the retinal thickness and the retinal structure.

[0053] In addition, the changes in the A wave and the B wave after the intravitreal injection were analyzed using electroretinogram (ERG) to evaluate the safety of the conditioned medium containing stem cell secretions. The conditioned medium containing stem cell secretions according to the present disclosure was injected into the vitreous of Wistar rats, and electroretinogram analysis was performed 28 days after the injection. The results are shown in FIG. 2. The A wave and the B wave of the electroretinogram after injection of the conditioned medium containing stem cell secretions were the same as those after injection of PBS, and no significant changes in the A wave and the B wave were observed, indicating that the intravitreal injection of the conditioned medium containing stem cell secretions did not cause damage to the function of the retina.

[0054] The above safety analysis demonstrated that the intravitreal injection of the conditioned medium containing stem cell secretions, including injection into the vitreous of animals, did not cause changes in the retinal thickness and the retinal structure, nor did it change the A wave and the B wave of the electroretinogram, indicating that the intravitreal injection of the conditioned medium containing stem cell secretions did not cause damage to the function of the retina, and thus was safe.

[0055] In subsequent observations, it was found that the above injection did not cause growth retardation, immune rejection, or death in rats, and thus the intravitreal injection of the conditioned medium containing stem cell secretions according to the present disclosure was safe.

[0056] Example 3: Effect of the pharmaceutical composition containing stem cell secretions on providing protection of retinal function

[0057] Using the Royal College of Surgeon rat (RCS rat) as an animal model, this study tested whether the conditioned medium containing stem cell secretions disclosed in this invention has the effect of delaying the rate of retinal degeneration in the rats.

[0058] First, rats with retinitis pigmentosa were injected intravitreally (IVI) with conditioned medium (CM) containing stem cell secretions and PBS, respectively, and electroretinography was performed 56 days after injection.

[0059] As shown in Figure 3, the electroretinography analysis revealed that injecting conditioned medium containing stem cell secretions into the vitreous humor 56 days after injection provided a protective effect on retinal function. The A and B waves in the RCS rat group were significantly higher than those in the PBS-injected group. * (p<0.05). Therefore, a single intravitreal injection of conditioned medium containing stem cell secretions can provide a slowing effect on retinal degeneration for at least two months.

[0060] Furthermore, as shown in Figure 4, after examining the retinal structure and thickness of RCS rats using optical coherence tomography, it was found that injecting conditioned medium containing stem cell secretions into the eye via vitreous injection could effectively increase retinal thickness.

[0061] Example 4: Protein Analysis of Pharmaceutical Compositions

[0062] The proteome analysis was performed on the conditioned medium containing stem cell secretome in the present disclosure, and the method of proteome analysis is a conventional technique in the art (e.g., Ding et al., Exp. Mol. Med. 2017 Dec 15; 49(12): e411), and a total of 818 proteins were identified, of which 124 proteins were secreted proteins. Further analysis of the expression levels showed that a total of 52 proteins had stable high expression levels in different batches of conditioned medium, and Figure 5 shows the 52 proteins and their interactions, and the interaction analysis was performed using the functional analysis tool STRING 9.0 for protein interaction network, and it was found that there were 10 proteins having protective effects on retinal degeneration, including pigment epithelium-derived factor (PEDF; also known as "serpin family F member 1 (serpin F1)"), tissue inhibitor matrix metalloproteinase 1 (TIMP-1), secreted protein acidic and rich in cysteine (SPARC; also known as "osteonectin"), alpha-2 Heremans Schmid glycoprotein (AHSG; also known as "fetuin A"), periostin (POSTN), decorin (DCN), retinoic acid receptor responder protein 2 (RARRES2; also known as "chemerin"), glia-derived nexin (GDN), sparc / osteonectin, cwcv, and kazal-like domains proteoglycan 1 (SPOCK1), and follistatin-like 1 (FSTL1) proteins.

[0063] Further, enzyme-linked immunosorbent assay (ELISA) was used to examine the absolute concentrations of the above-mentioned 10 proteins in the conditioned media of three different production batches to evaluate the performance stability of the functional proteins in the conditioned media of different batches. As shown in FIG. 6, the concentrations of the 10 proteins in the conditioned media of three different production batches were found to be at least 60 pg / mL.

[0064] Further, the intraocular half-lives of PEDF, SAPRC, and POSTN were analyzed, which is a known method in the art (e.g., Daniele et al., BMJ Open Ophthalmol. 2022 Sep; 7(1): e000981). The results showed that the half-life of PEDF was 43.2 to 51.5 hours, the half-life of SPARC was 26.7 to 34.3 hours, and the half-life of POSTN was 21.2 to 36.3 hours, indicating that the proteins are not easily released outside the eye in the vitreous body, and the slow release of the drug in this viscous environment can prolong the half-life of the protein in the body.

[0065] Example 5: Proteins in pharmaceutical compositions have retinal protection function

[0066] The retinal protection function of each protein contained in the above-mentioned pharmaceutical composition was investigated, and it was found that each protein alone can provide retinal protection function.

[0067] For example, after treating human retinal pigment epithelial cells with PEDF or POSIN under high oxidative stress, it was found that PEDF and POSIN can provide an anti-oxidative stress effect on retinal pigment epithelial cells, thereby reducing the proportion of cell death and thus having the ability to protect the retina from degeneration. The high oxidative stress experiment was performed according to the known method in the art, such as Wang et al., Mol. Med. Rep. 2019 Jan; 19(1): 59-74. Specifically, human retinal pigment epithelial cell line ARPE-19 was used, with 3,000 cells per well seeded in a cell culture dish, cultured with 1% FBS, and then PEDF or POSIN protein was added to the cell culture medium after 21 hours. After 3 hours, H2O2 (1.5 mM) was added for high oxidative stress test, and cell survival was evaluated by CCK-8 cell survival rate assay 24 hours after the addition of H2O2.

[0068] Figure 7 shows the results of the aforementioned hyperoxidative stress test on human retinal pigment epithelial cells ARPE-19 using H2O2 and treatment with PEDF or POSIN protein, showing that treatment with different concentrations of PEDF (31.25 ng / mL to 500 ng / mL) or POSIN (12.5 ng / mL to 200 ng / mL) greatly improved cell survival under hyperoxidative stress as determined by CCK-8 cell survival rate compared to the untreated control group.

[0069] In addition, the protective effect of the aforementioned proteins on mitochondrial aging and degeneration of retinal pigment epithelial cells was also analyzed. Specifically, the sodium iodate (NaIO3) was used to induce oxidative stress and mitochondrial superoxide degeneration in retinal pigment epithelial cells, and the effect of PEDF, POSIN and SPARC on inhibiting oxidative stress and mitochondrial superoxide in human retinal pigment epithelial cell line ARPE-19 was tested. The method for detecting oxidative stress is a known technique in the art, such as Lee, J.-J. et al., Oxidative Medicine and Cellular Longevity 2022, e1792894 (2022) and Hsu, M.-Y. et al., Antioxidants 10, 1125 (2021).

[0070] In the experiment, 12,000 cells per well were seeded in an 8-well cell culture slide and cultured with 1% FBS. After 21 hours, PEDF (250 ng / mL), POSIN (200 ng / mL) or SPARC (200 ng / mL) protein was added to the cell culture medium, and after 3 hours, NaIO3 (6 mM) was added for mitochondrial superoxide test. JC-1 was used for cell staining 24 hours after the addition of NaIO3 to evaluate the degree of mitochondrial superoxide. JC-1 is a positively charged dye whose staining principle is related to the membrane potential of mitochondria. In normal polarized mitochondria, the dye accumulates in the inner membrane to form JC-1 aggregates and emits red light (585 / 590 nm). In depolarized mitochondria of apoptotic cells, when the membrane potential is less than 100 mV, the dye flows out of the mitochondria into the cytoplasm, forming JC-1 monomers and emitting green light (510 / 527 nm). Therefore, the green / red light ratio can be used to observe the apoptosis of cells.

[0071] As shown in Fig. 8, it can be found from the staining results of JC-1 that the three efficacy proteins PEDF, POSIN and SPARC can effectively reduce the degree of mitochondrial superoxide, have a protective effect on the aging and degeneration of human retinal pigment epithelial cells induced by sodium iodate, and can greatly reduce the mitochondrial depolarization of human retinal pigment epithelial cells. Among them, PEDF has the best protective effect on the aging and degeneration of human retinal pigment epithelial cells.

[0072] The above examples are used to describe the present disclosure, and those skilled in the art should understand that other modifications and variations are possible without departing from the scope of the present disclosure. Therefore, the scope of protection of the present disclosure should be defined by the appended claims.

Claims

1. A pharmaceutical composition comprising stem cell secretome and a pharmaceutically acceptable excipient, characterized in that, The stem cell secretion comprises at least one protein from the group consisting of pigment epithelium-derived factor, tissue inhibitor of metalloproteinase 1, cysteine-rich acidic secretory protein, alpha 2-HS glycoprotein, periostin, decorin, retinoic acid receptor response protein 2, glia-derived nexin, bone morphogenetic protein 1, and follistatin-like protein 1, or any combination thereof.

2. The pharmaceutical composition according to claim 1, wherein The stem cell is a mesenchymal stem cell.

3. The pharmaceutical composition according to claim 2, wherein The mesenchymal stem cell is an adipose-derived mesenchymal stem cell.

4. The pharmaceutical composition according to claim 1, wherein The stem cell is cultured in vitro.

5. The pharmaceutical composition according to claim 4, wherein The stem cell secretion is present in a conditioned medium of the stem cell cultured in vitro.

6. The pharmaceutical composition according to claim 1, wherein The stem cell secretion comprises at least two proteins from the group consisting of pigment epithelium-derived factor, tissue inhibitor of metalloproteinase 1, cysteine-rich acidic secretory protein, alpha 2-HS glycoprotein, periostin, decorin, retinoic acid receptor response protein 2, glia-derived nexin, bone morphogenetic protein 1, and follistatin-like protein 1.

7. The pharmaceutical composition according to claim 6, wherein The stem cell secretion comprises cysteine-rich acidic secretory protein.

8. The pharmaceutical composition according to claim 7, wherein The stem cell secretion comprises pigment epithelium-derived factor, periostin, and cysteine-rich acidic secretory protein.

9. The pharmaceutical composition of claim 1, which is a cell-free composition.

10. Use of a pharmaceutical composition for the preparation of a medicament for the treatment of degenerative ocular diseases, characterized in that, The pharmaceutical composition comprises stem cell secretion and a pharmaceutically acceptable excipient, wherein the stem cell secretion comprises at least one protein from the group consisting of pigment epithelium-derived factor, tissue inhibitor of metalloproteinase 1, cysteine-rich acidic secretory protein, alpha 2-HS glycoprotein, periostin, decorin, retinoic acid receptor response protein 2, glia-derived nexin, bone morphogenetic protein 1, and follistatin-like protein 1.

11. Use according to claim 10, characterized in that, The pharmaceutical composition is injected into the vitreous for administration to a subject in need thereof.

12. Use according to claim 11, characterized in that, The pharmaceutical composition delays retinal degeneration in the subject.

13. Use according to claim 12, characterized in that, The delay of retinal degeneration lasts at least one month.

14. Use according to claim 11, characterized in that, The pharmaceutical composition protects retinal function in the subject.

15. Use according to claim 14, characterized in that, The protection of retinal function lasts at least one month.

16. Use according to any one of claims 10 to 15, characterized in that, The degenerative ocular disease is retinal degeneration, retinitis pigmentosa, glaucoma, or macular degeneration.

Images