PEDF-derived peptides and their use to promote meibomian gland regeneration

Abstract

A pharmaceutical composition or method for promoting meibomian gland regeneration or treating / preventing dry eye syndrome comprises administering to a subject in need thereof a pharmaceutical composition comprising a PEDF-derived short peptide (PDSP) or a variant of PDSP, wherein PDSP comprises residues 93-106 of human pigment epithelium-derived factor (PEDF).

Description

[Technical Field]

[0001] This invention relates to PEDF-derived peptides and their use in meibomian gland regeneration or the treatment of dry eye. [Background technology]

[0002] Meibomian gland dysfunction (MGD) is characterized by a decrease in the quantity and / or quality of meibomian gland secretions, instability of the tear film lipid layer, and symptoms of eye irritation. 1~3 MGD accounts for two-thirds of all cases of dry eye disease (DED), and is therefore considered a growing public problem, especially among the elderly. 1、2 However, current clinical treatments for MGD, including topical medications, meibomian gland (MG) expression, Lipiflow, and intense pulsed light (IPL) therapy, are mostly symptomatic, as they often aim primarily to alleviate the symptoms of DED and prevent further MG atrophy, rather than directly addressing the underlying etiology of MGD. 1、2、4 .

[0003] There are three forms of MGD: hypersecretion-type MGD, hyposecretion-type MGD, and obstructive MGD. 5 Obstructive MGD is considered the most common type and is thought to be involved in hyperkeratosis of the ductal opening, leading to ductal obstruction and subsequent acinar atrophy. 3、5 However, the anterior displacement of the mucocutaneous junction in MGD patients and the findings of non-keratinized ductal epithelial cells at the openings of mouse MG do not support the conventional theory of hyperkeratosis as the primary mechanism of MGD. 5、6 In age-related meibomian gland disease (MGD), glandular atrophy accompanied by decreased cell proliferation was observed in both human and mouse meibomian glands. 7、8 Acinar tissue atrophy may be a major etiology, leading to an imbalance between lipids and duct cells, or changes in the lipid / protein ratio that contribute to occlusion. 5、9 . [Overview of the Initiative] [Problems that the invention aims to solve]

[0004] Meibomian gland dysfunction (MGD) affects many patients, but current treatments are mostly symptomatic. Therefore, more effective treatments for MGD are needed. [Means for solving the problem]

[0005] Embodiments of the present invention relate to a method for promoting meibomian gland regeneration and a method for treating dry eye using short-chain peptides derived from pigment epithelial factor (PEDF).

[0006] One aspect of the present invention relates to a method for promoting meibomian gland regeneration. A method according to one embodiment of the present invention comprises administering to a subject in need of such regeneration a pharmaceutical composition comprising a PEDF-derived short-chain peptide (PDSP) or a variant of PDSP, wherein the PDSP comprises residues 93 to 106 of human pigment epithelial-derived factor (PEDF).

[0007] One aspect of the present invention relates to a method for treating dry eye syndrome. A method according to one embodiment of the present invention comprises administering to a subject in need of such treatment a pharmaceutical composition comprising a PEDF-derived short-chain peptide (PDSP) or a variant of PDSP, wherein the PDSP comprises residues 93 to 106 of human pigment epithelium-derived factor (PEDF).

[0008] Other aspects of the present invention will become apparent from the following detailed description and the appended claims. [Brief explanation of the drawing]

[0009] [Figure 1]It shows MG atrophy and instability of the tear film in aged mice. Panel (A) shows the upper and lower eyelids collected from young and aged mice stained with Oil-Red-O (ORO). Myoepithelial cells (red) were observed in the main ducts (arrow) and acini (arrowhead). Representative images were obtained from 7 eyelids of aged mice and 6 eyelids of young mice. Panel (B) shows the classification of meibomian gland atrophy that was calculated and converted to an MG score based on the percentage of the atrophy area. Panel (C) shows frozen sections of the upper eyelids of aged and young mice stained with ORO. Panel (D) shows the tear film break-up time evaluated from 16 eyes of 8 young mice and 12 eyes of 6 aged mice. Data are reported as mean ± SE. *P < 0.05 vs. young mice. **P < 0.001 vs. young mice.

[0010] [Figure 2] It shows PEDF expression in the upper eyelids of young and aged mice. Representative cross-sections of PEDF staining showed acini in young mice (A) and aged mice (B). Immunostaining with only the secondary antibody was used as a negative control (C). The box in the low magnification image (upper panel) indicates the position of the high magnification image (lower panel), and the high magnification image (lower panel) shows that PEDF is mainly expressed in the nuclei of progenitor cells (black arrows; (D) and (E)). PEDF expression was observed throughout the acini and was even stronger in the cytoplasm of the acinar base. (F) The histopathological score of PEDF was calculated based on staining intensity, intensity of the cytoplasm of the acinar base, and the percentage of PEDF-positive cell nuclei. For analysis, 3 randomized images were obtained from each eyelid. Representative images were obtained from 6 eyelids of 6 different mice in each group (original magnification: X400). *P < 0.05 vs. young group. Scale bar, 50 μm.

[0011] [Figure 3]29-mer promotes the proliferation of preadipocytes in aged mice. To detect DNA synthesis, BrdU was intraperitoneally injected immediately after 29-mer treatment, and the eyelids were harvested at 24 h. Under normal conditions (no treatment), aged mice (A) showed fewer BrdU-positive cells than young mice (D). 29-mer increased BrdU-positive cells at the acinar basal in both aged mice (C) and young mice (F), while DMSO showed no effect (B and E). The red dashed circles indicate the central ducts. The black arrows indicate BrdU-positive progenitor cells. (G) The PEDF treatment effect was calculated based on BrdU-positive cells in the acinar basal. For analysis, three randomized images were obtained from each eyelid. Representative images were obtained from six eyelids of six different mice in each group (original magnification: X400). Representative images were obtained from three eyelids of three different mice in each group (original magnification: X400). #P < 0.05 vs. normal condition of young mice, *P < 0.05 vs. aged mice treated with DMSO or 18-mer. **P < 0.05 vs. normal condition of young mice, or young mice treated with vehicle DMSO. Scale bar, 50 μm.

[0012] [Figure 4] Shows cell proliferation in the upper eyelid 5 days after treatment. After a single subconjunctival injection, BrdU was intraperitoneally injected on day 0 and day 3. Then, the eyelids were harvested on day 5. (A) and (C) show the controls by DMSO treatment for aged mice and young mice, respectively. (C) and (D) show the PDSP treatment for aged mice and young mice, respectively. BrdU-positive cells were mainly acinar progenitor cells (black arrows), and a small number of meibomian gland epithelial cells (meibocytes) were positive for BrdU staining (D, red arrows). (E) The PEDF treatment effect was calculated based on BrdU-positive cells in the acinar basal. For analysis, three randomized images were obtained from each eyelid. Representative images were obtained from three eyelids of three different mice in each group (original magnification: X400). *P < 0.001 vs. aged mice treated with DMSO. Scale bar, 50 μm.

[0013] [Figure 5] Immunohistochemical analysis of stem cell marker p63 expression in the upper eyelid 5 days after single treatment is shown. (A) and (B) show baseline p63 expression in aged and juvenile mice, respectively. (C) and (D) show DMSO and PDSP treatment in aged mice, respectively. The red dashed circle indicates the central duct. (E) The number of p63-positive cells per acinar was assessed. Representative images were obtained from three eyelids of three different mice in each group (original magnification: X400). Three randomized images were obtained from each eyelid for analysis. *P<0.001 vs juvenile mice, **P<0.001 vs aged mice treated with DMSO. Scale bar, 50 μm.

[0014] [Figure 6] The study shows that the 29-mer enhances tear film stability in aged mice. Levels of tear film breakdown time (TBUT) (A) and tear film secretion (B) are shown at 1, 2, 3, 4, and 8 weeks after 29-mer injection. Values ​​are expressed as mean ± SE. Data were obtained from six eyelids of six different mice in each group. Three measurements of tear film breakdown time obtained from one eyelid were recorded. *P<0.05 vs. vehicle group at the same time point.

[0015] [Figure 7] The 29-mer is shown to increase acinar size in aged mice. (A) Left, ORO staining of whole tissue specimens of the upper eyelid. Right, MG area analyzed using the area calculation tool in Adobe Photoshop 7.0. (B) shows a histogram of upper eyelid MG size measured by Photoshop, expressed in pixels. Frozen sections of the upper eyelid were stained with ORO. Histogram of acinar size measured by Adobe Photoshop 7.0, expressed in pixels. Representative images of whole tissue specimens were obtained from seven eyelids of seven different mice in each group. Representative images of frozen sections were obtained from three eyelids of three different mice in each group. *P<0.05 vs. vehicle group.

Mode for Carrying Out the Invention

[0016] Embodiments of the present invention relate to a method for promoting meibomian gland regeneration using a PEDF-derived short-chain peptide (PDSP). The meibomian gland is a holocrine exocrine gland. The meibomian gland is located at the edge of the eyelid within the tarsal plate and is responsible for supplying meibum, an oily substance that prevents evaporation of the tear film of the eye. Meibomian gland dysfunction (MGD) is the most common cause of dry eye syndrome (or dry eye disease). MGD can cause inflammation of the eyelid, called blepharitis, especially along the edges.

[0017] In normal MG homeostasis, meibomian gland epithelial cells within the MG acini continuously differentiate from stem cells within the basal cell layer surrounding the acini. 4 . In the present specification, the inventors have first found that the PEDF protein is mainly expressed in the nuclei of acinar basal cells (progenitor cells) and the cytoplasm of acinar bases. Along with the changes of aging, the expression of the PEDF protein was significantly decreased.

[0018] Pigment epithelium-derived factor (PEDF) is a secreted glycoprotein containing 418 amino acids and having a molecular weight of about 50 kDa. PEDF is a multifunctional protein that was first identified and isolated from the culture supernatant of cultured human fetal retinal pigment epithelial cells. 10~12 . PEDF is widely expressed in the liver, adipose tissue, eye, heart, and pancreas and plays a fundamental role in the organogenesis and maintenance of homeostasis of adult tissues. 12~14 .

[0019] Different motifs of PEDF exhibit different biological activities. For example, the 44-mer motif (amino acid positions Val 78 ~Thr 121 ) determines the neurotrophic activity and cell division activity of PEDF. 12、15 . On the other hand, the 34-mer fragment (residues 44 - 77 of PEDF) has been identified to have anti-angiogenic activity. The inventors have found that the 44-mer (Val 78 ~Thr 121) discovered that it can induce the proliferation and regeneration of stem cells in the limbus of rabbit corneas. 16~18 Furthermore, there is a shorter peptide 29-mer (residue Ser 93 ~Thr 121 ) is myogenic stem cell and C2C 12 It was found to induce myoblast proliferation. 15 This invention was based on the discovery that PEDF protein expression in the MG acinars decreases with age.

[0020] The inventors of this invention unexpectedly discovered that certain PEDF-derived short-chain peptides (PDSPs) can increase the proliferation of acinar progenitor cells, as well as acinar size and tear film stability in vivo. These PDSPs can promote meibomian gland regeneration and can be used to treat or prevent dry eye disease.

[0021] The PDSP of the present invention is based on human PEDF residues 93-121 ( 93 SLGAEQRTESIIHRALYYDLISSPDIHGT 121 Based on the peptide region corresponding to Sequence ID No. 1), the inventors identified serine-93, alanine-96, glutamine-98, isoleucine-103, isoleucine-104, and arginine-106 as important for activity, as evidenced by the significant loss of activity when these residues were individually substituted with alanine (or alanine-96 with glycine). In contrast, alanine (or glycine) substitutions of other residues in the 29-mer did not clearly alter the activity, suggesting that PDSP variants having amino acid substitutions (particularly homologous amino acid substitutions) in these other residues (i.e., residues 94, 95, 97, 99-102, 105, and 107-121) could also be used to prevent and / or treat osteoarthritis or to induce chondrogenesis.

[0022] These results indicate that the core peptide containing the antinociceptive effect is located at residues 93-106. 93 SLGAEQRTESIIHR106 This indicates that it is located in the region containing (SEQ ID NO: 2). Therefore, the shortest PDSP peptide with antinociceptive activity may be a 14-mer. Those skilled in the art will understand that the addition of additional amino acids to this core peptide at the C-terminus and / or N-terminus does not affect this activity. That is, the PDSP of the present invention may be any peptide containing residues 93-106 of human PEDF. Therefore, the PDSP peptide of the present invention may be a 14-mer, 15-mer, 16-mer, etc., including the 29-mer used in the experiment.

[0023] Furthermore, as described above, substitutions within these short-chain peptides can retain activity as long as the key residues (serine-93, alanine-96, glutamine-98, isoleucine-103, isoleucine-104, and arginine-106) are conserved. In addition, mouse variants (having two substitutions, histidine-98 and valine-103, compared to the human sequence) are also active. The corresponding mouse sequences are the mo-29mer (SLGAEHRTESVIHRALYYDLITNPDIHST, SEQ ID NO: 3) and the mo-14mer (SLGAEHRTESVIHR, SEQ ID NO: 4). Therefore, the general sequence of the active core is ( 93 SXXAXQ / HXXXXI / VIXR 106 (X represents any amino acid residue); Sequence ID No. 5). Some examples of PDSP sequences that may be used with embodiments of the present invention are shown in the table below (position numbering is based on the position of the 14-mer). These examples are not intended to be limiting. [Table 1] TIFF0007883831000002.tif228166TIFF0007883831000003.tif229166TIFF0007883831000004.tif49166

[0024] The PDSP peptides of the present invention can be chemically synthesized or expressed using a protein / peptide expression system. These PDSP peptides can be used in pharmaceutical compositions for the prevention and / or treatment of osteoarthritis. The pharmaceutical compositions may contain any pharmaceutically acceptable excipients, and the pharmaceutical compositions can be formulated in forms suitable for administration such as topical, oral, or injection. Various formulations for such applications are known in the art and can be used in conjunction with embodiments of the present invention.

[0025] The PEDF derivative of the present invention, for example, the 29-amino acid PDSP 29-mer, stimulates the proliferation and lipid synthesis of acinar progenitor cells, as evidenced by the higher number of p63-positive basal cells and better staining of whole tissue and frozen sections by Oil Red O (ORO) in PDSP-treated aged mice compared to untreated mice. The 29-mer also improved tear film stability in aged mice.

[0026] The results described in this invention indicate that PEDF is expressed in greater quantities in undifferentiated acinar progenitor cells than in differentiated meibomian gland epithelial cells. In aged mice, PEDF protein expression within MG was decreased, and the cell cycle and p63 labeling of acinar progenitor cells were significantly reduced. Other results indicate that levels of PEDF expression decreased with age in the choroid / RPE complex and skin. The decrease in PEDF protein in various tissues during the normal aging process may be important for age-related diseases.

[0027] Our tests also showed that direct injection of PDSP (e.g., 29-mer) into young and aged mice resulted in proliferation of basal acinar cells at 24 hours. On day 5, a significant difference in cell proliferation was observed between 29-mer injection and DMSO injection in aged mice, but not in young mice. In young mice, endogenous PEDF levels are higher than in aged mice, and the addition of 29-mer may lead to reaching steady-state PEDF concentrations and receptor occupancy. Therefore, no significant increase was detected in young mice.

[0028] Meibomian glands are modified sebaceous glands with total secretory differentiation. Sebaceous gland cell differentiation is strongly associated with enhanced intracellular lipid synthesis and accumulation. Our tests revealed that PDSP not only exerts a promitotic effect on acinar progenitor cells but also enhances acinar differentiation. PPARγ signaling may be involved in the PEDF-mediated lipid synthesis signaling pathway. Therefore, PEDF may promote acinar differentiation by regulating PPARγ.

[0029] MGD has been shown to be associated with inflammatory cytokines IL-1α and mature IL-1β on the ocular surface. 37 PEDF is known for its anti-inflammatory activity. 12 PEDF has been shown to block IL-1β by suppressing the activation of the inflammatory mediator c-Jun N-terminal kinase in human hepatocytes. 40 Therefore, PEDF may improve symptoms in MGD patients by improving inflammatory proteins on the ocular surface. The results presented herein indicate that the 29-mer does not affect tear secretion, but can increase lipid production and tear film stability, as evidenced by the increase in TBUT.

[0030] In summary, the results reported herein indicate that PEDF peptide derivatives can promote the proliferation of acinar progenitor cells. Direct stimulation of acinar progenitor cell proliferation, and improvement in lipid production and tear film stability in vivo, suggest that PEDF peptide derivatives may be a potential therapeutic agent for MGD.

[0031] Embodiments of the present invention are illustrated by the following specific examples. In these specific examples, the 29-mer (SEQ ID NO: 1) is used. However, other PDSPs (e.g., the 14-mer, SEQ ID NO: 2, or SEQ ID NO: 3) can also be used to achieve the same results. Those skilled in the art will understand that these examples are for illustrative purposes only and can be modified and altered without departing from the scope of the present invention.

[0032] Chemicals and antibodies The antibodies used in this study were anti-PEDF antibody (sc-25594, Santa Cruz Biotechnology, CA), BrdU (GTX42641, GeneTex, San Antonio, TX), and p63 (mab4135, Millipore, Billerica, MA). (29-mer (Ser)) 93 ~Thr 121 ) and 18-mer (Glu 97 ~Ser 114 A control peptide was synthesized and modified for stability by acetylation of the NH2 terminus and amidation of the COOH terminus. It was then characterized by mass spectrometry (purity >90%) using GenScript (Piscataway, NJ).

[0033] Animals and treatments C57BL / 6 mice aged 12–15 months and C57BL / 6 mice aged 4–8 months were used. These mice were kept in a standard, pathogen-free environment at 24°C ± 1°C and 60% ± 10% relative humidity. All procedures were approved by the Mackay Memorial Hospital Review Board for animal studies and were carried out in accordance with the ARVO statement for the Use of Animals in Ophthalmic and Vision Research. Mice were anesthetized by intraperitoneal injection of a mixture of zoletil (6 mg / kg) and xylazine (3 mg / kg). One drop of 0.5% propalacaine hydrochloride (Alcaine; Alcon, Fort Worth, TX, USA) was administered before any ophthalmic surgery.

[0034] The 29-mer was reconstituted in DMSO to a final concentration of 100 μM. Separate doses of 10 μl of the 29-mer (100 μM) mixed with 90 μl of phosphate-buffered saline (PBS) were injected into the upper and lower conjunctival fornix. 10 μl of DMSO mixed with 90 μl of PBS was used as a control. To evaluate the effects of the 29-mer on tear film breakup time (TBUT) and tear secretion in aged mice, subconjunctival injections of the 29-mer were introduced weekly for up to one month, followed by two months of follow-up.

[0035] At one month, upper eyelid tissue samples were provided for Oil Red O (ORO) staining of all tissue specimens. The size of MG tissue within all tissue specimens was quantified in pixels using color range selection and histogram tools with a computer-aided image analyzer (Adobe Photoshop 7.0).

[0036] Tear film breakdown time To avoid decreased lipid secretion caused by the absence of blinking under prolonged anesthesia, the tear film breakdown time (TBUT) was measured immediately after anesthetizing the mice. 19、20The procedure involved dropping 1.5 μL of 0.1% topical fluorescein (Fluor-I-Strip; Ayerst Laboratories, Philadelphia, PA, USA) onto the ocular surface. After three forced blinks, TBUT was recorded in milliseconds under a slit lamp equipped with a blue-free barrier filter. Three measurements were obtained from each eye. TBUT was then taken by a single ophthalmologist, blinded to the treatment group, in a standard environment at the same time of day (2-3 p.m.).

[0037] Eyelid whole tissue specimen After removing body hair, fresh mouse eyelids were collected, immediately fixed overnight with 4% paraformaldehyde, and rinsed with PBS. 4 An Oil-Red-O (ORO) solution was prepared by mixing the stock solution (300 mg of ORO powder in 100 ml of 99% isopropanol solution) and filtered. Eyelids were immersed in 60% 2-propanol for 15 minutes, stained with the ORO solution for 30 minutes, and then destained with 60% 2-propanol for 15-20 minutes to achieve optimal lipid staining. 4 Next, the eyelids were mounted and photographed using a microscope.

[0038] Meiboscale Meibography images were used to classify MG in young and aged mice according to the Meiboscale. 21 In summary, MG atrophy was indicated as follows: Grade 0 for no missing area, Grade 1 for <25% missing area, Grade 2 for 25%-50% missing area, Grade 3 for 51%-75% missing area, and Grade 4 for 75% missing area. The MG scores were analyzed as follows: Grade 0, 5; Grade 1, 4; Grade 2, 3; Grade 3, 2; Grade 4, 1.

[0039] Oil red O for lipids Eyelid tissue was embedded in OCT and sectioned into 8 μm thick sections. Frozen sections were immersed in 60% 2-propanol for 1 minute, stained with filtered ORO solution for 15 minutes, rinsed with PBS, and counterstained with hematoxylin. 4 .

[0040] 5-bromo-2'-deoxyuridine incorporation assay Following subconjunctival injection of a 29-mer or DMSO, 0.1 mg / g body weight of 5-bromo-2'deoxyuridine (BrdU) was administered intraperitoneally. Upper eyelids were collected at 24 hours to evaluate the proliferation of acinar progenitor cells. To test cell mitosis, mice were intraperitoneally injected with BrdU daily for 3 days, and upper eyelids were collected on day 5. Slides were treated with 1N HCl at 95°C for 20 minutes before immunohistochemical analysis of BrdU.

[0041] Measurement of tear volume Tear volume was measured using a phenol red cotton thread tear test with ZONE-QUICK cotton thread (Yokota, Tokyo, Japan). 19、20 After general anesthesia, the lower eyelid was slightly pulled down, and a 1 mm portion of cotton suture was placed on the conjunctiva at a distance of one-third from the outer corner of the eye. Each eye was tested for one minute with the eyes open. The red portion of the cotton suture was measured in millimeters.

[0042] Immunohistochemical examination Immunohistochemical testing (IHC) was performed as described above, and the modified results were... 22Formalin-fixed, paraffin-embedded mouse specimens were deparaffinized in xylene and rehydrated with a stepwise series of ethanol concentrations. Slides were blocked with 10% goat serum for 60 minutes, then incubated overnight at 4°C with primary antibodies against BrdU (1:800 dilution), PEDF (1:50), or p63 (1:200). Subsequently, slides were incubated with appropriate peroxidase-labeled goat immunoglobulin (1:500 dilution; Chemicon, Temecula, CA) for 20 minutes, then incubated with chromogenic substrate (3,3-diaminobenzidine) for 2 minutes, and counterstained with hematoxylin. Quantification was estimated based on high-quality images acquired using a Pannoramic digital slide scanner (3Dhistech Ltd., Budapest, Hungary).

[0043] PEDF staining classification PEDF expression was classified as follows: (A) Weak staining of the entire acinar: 0; Strong staining of the entire acinar: 1; (B) No tendency for stronger staining in the basal cytoplasm than other areas of the same acinar: 0; Weak but stronger staining of the basal acinar cytoplasm than other areas: 1; Moderate but stronger staining of the basal acinar cytoplasm than other areas: 2; Strong staining of the basal acinar cytoplasm than other areas: 3; (C) Not expressed in the basal cell nucleus: 0; <50% of basal cell nuclei stained positively for PEDF: 1; >50% of basal cell nuclei stained positively for PEDF: 2. The (A)+(B)+(C) scores were summed, and the total score may range from 0 to 6.

[0044] statistical analysis Results were expressed as mean ± standard deviation. Statistical analysis was performed using SPSS version 18.0 (SPSS Inc., Chicago, IL, USA). The Mann-Whitney test was used for statistical comparisons. Values ​​of P < 0.05 were considered statistically significant.

[0045] Myelomyelopathy atrophy in aged mice In aged mice, a reduction in MG acinar size was observed in all tissue specimens stained with ORO (Figure 1A). The MG scores for the upper eyelids of juvenile and aged mice were 4±0.82 and 2.5±0.63 (P=0.015), respectively. The MG scores for the lower eyelids of juvenile and aged mice were 2.5±0.84 and 1.8±0.84 (P=0.006), respectively (Figure 1B). Findings in eyelid cross-sections stained with ORO correlated with morphological changes detected in all tissue specimens (Figure 1C). In aged mice, a reduction in acinar size around the central duct was observed in frozen sections stained with ORO. The tear film breakdown time (TBUT) in aged mice was 317.36 ± 119.76, which was significantly shorter than that of juvenile mice (389.04 ± 49.18) (P<0.001) (Figure 1D). These results indicate that aged mice have significant MG atrophy and tear film instability.

[0046] Decreased PEDF protein expression in MG acinars of aged mice To observe the distribution of PEDF protein throughout the acini, a cross-section of the acini of the upper eyelid was taken. 4 This was tested by IHC. The results showed that PEDF was expressed in the nuclei of progenitor cells or in early differentiated meibomian gland epithelial cells near progenitor cells (Figure 2A, B, D, and E). Furthermore, the intensity of PEDF expression was stronger in the cytoplasm of the acinar basal cytoplasm than in the proximal end near the ductal tissue (Figure 2D). The tendency for higher PEDF protein expression in the acinar basal cytoplasm was less pronounced in aged mice (Figure 2E). In aged mice, the intensity of PEDF expression in the acinar tissue, including the nuclei of progenitor cells, was significantly reduced compared to juvenile mice. The overall score of PEDF protein expression, shown in Figure 2F, was reduced in aged mice compared to juvenile mice (3.17±0.83 vs. 4.72±1.04, P<0.001).

[0047] PEDF peptide promotes the proliferation of acinar progenitor cells. A decrease in the cell cycle of MG acinars was observed in aged mice. 7To evaluate the promitotic effect of PDSP on MG acinar size, mice were intraperitoneally injected with BrdU and euthanized 24 hours after treatment. At 24 hours, all BrdU-positive cells were found to be acinar progenitor cells located at the base of the acinars. In the untreated group, younger mice had more BrdU-positive cells per acinar compared to older mice (Figure 3A and B, 1.44±0.40 vs. 0.73±0.21, P=0.001). The proliferation rate in the untreated group was similar to that in the DMSO-treated group in either young or older mice (Figure 3A, B, D, and E). The 29-mer peptide was able to increase the number of proliferating cells in younger mice compared to DMSO (Figure 3F, 2.35±0.73 vs. 1.68±0.71, P=0.041). Furthermore, PDSP-treated aged mice showed an increased number of BrdU-positive cells compared to the DMSO group (Figure 3C, 1.67±0.58 vs. 0.74±0.34 cells / acinel, P=0.002). The control peptide 18-mer did not affect cell proliferation in aged mice (Figure 3G, 1.00±0.39, p=0.156 compared to DMSO).

[0048] To evaluate the effect on MG homeostasis, mice were intraperitoneally injected with BrdU for 3 days and euthanized on day 5. BrdU pulsed labeling assays showed a continuous increase in cell proliferation from 24 hours to day 5, with PDSP-treated aged mice showing higher proliferation than DSMO-treated aged mice (Figure 4A, B, and E, 4.29±1.19 vs. 2.24±0.50, P<0.001). In contrast, at day 5, there was no difference in cell proliferation between PDSP-treated and DSMO-treated aged mice (Figure 4C and D, 6.70±1.35 vs. 5.78±1.84, P=0.233). Notably, at day 5, some meibomian gland epithelial cells were positive for BrdU staining (Figure 4D). The above results indicate that the 29-mer that promoted the proliferation of acinar progenitor cells completely blocked the differentiation of meibomian gland epithelial cells.

[0049] Furthermore, the inventors investigated the number of acinar progenitor cells using p63 as a marker.4 The number of p63-expressing cells was significantly reduced in aged mice compared to juvenile mice (Figure 5A and B, 6.51±1.48 vs. 10.21±0.98, P<0.001). The 29-mer increased the number of p63-expressing cells in aged mice to a level comparable to that of juvenile mice (Figure 5D, 10.98±2.75). Furthermore, DMSO did not show any effect on the increase of acinar progenitor cells (Figure 5C, 7.06±1.9, P<0.001 compared to the 29-mer).

[0050] PEDF peptides improve TBUT and lipid synthesis. To evaluate the effect of the 29-mer on lipid (meibum) formation, subconjunctival injections of the 29-mer were administered weekly for up to 4 weeks. TBUT and phenol red cotton thread tear secretion tests were performed at weeks 1, 2, 3, 4, and 8 (Figure 6A). TBUT in mice treated with the 29-mer was significantly longer than in the control group from week 1 to week 4. The difference remained statistically significant up to week 8. Tear secretion tests were invariant between the two groups (Figure 6B). These results suggest that the increase in TBUT was due to an improvement in the lipid layer.

[0051] Furthermore, we examined the lipids secreted by MG. Lipid (meibum) production by MG in the upper eyelid was evaluated using whole eyelid tissue specimens stained with ORO. After 29-mer treatment, an increase in MG acinar size was observed compared to DMSO (Figure 7A, 2347530±34986.4 vs. 1921689±299347.1 pixels / eyelid, P=0.048). In PDSP-treated mice, ORO-stained eyelid cross-sections showed relatively overall ORO staining within the cytoplasm of differentiating meibomian gland epithelial cells (Figure 7B).

[0052] Although the above examples use a 29-mer to illustrate embodiments of the present invention, the active core peptide is a 14-mer. As described above, alanine scanning identifies essential residues within the 14-mer, while substitutions are permitted for non-essential residues. These other variants of PDSP may also be used in conjunction with embodiments of the present invention.

[0053] Embodiments of the present invention have been described using a limited number of examples. Those skilled in the art will understand that modifications and alterations are possible without departing from the scope of the invention. Accordingly, the scope of the invention should be limited only by the accompanying claims.

[0054] References 1.Finis D, Hayajneh J, Konig C, Borrelli M, Schrader S, Geerling G. Evaluation of an automated thermodynamic treatment (LipiFlow(R)) system for meibomian gland dysfunction: a prospective, randomized, observer-masked trial. Ocul Surf 2014;12(2):146-154. 2.Liu R, Rong B, Tu P, et al. Analysis of Cytokine Levels in Tears and Clinical Correlations After Intense Pulsed Light Treating Meibomian Gland Dysfunction. Am J Ophthalmol 2017;183:81-90. 3.Knop E, Knop N, Millar T, Obata H, Sullivan DA. The international workshop on meibomian gland dysfunction: report of the subcommittee on anatomy, physiology, and pathophysiology of the meibomian gland. Invest Ophthalmol Vis Sci 2011;52(4):1938-1978. 4.Reneker LW, Wang L, Irlmeier RT, Huang AJW. Fibroblast Growth Factor Receptor 2 (FGFR2) Is Required for Meibomian Gland Homeostasis in the Adult Mouse. Invest Ophthalmol Vis Sci 2017;58(5):2638-2646. 5.Jester JV, Parfitt GJ, Brown DJ. Meibomian gland dysfunction: hyperkeratinization or atrophy? BMC Ophthalmol 2015;15 Suppl 1:156. 6.Ong BL, Hodson SA, Wigham T, Miller F, Larke JR. Evidence for keratin proteins in normal and abnormal human meibomian fluids. Curr Eye Res 1991;10(12):1113-1119. 7.Nien CJ, Paugh JR, Massei S, Wahlert AJ, Kao WW, Jester JV. Age-related changes in the meibomian gland. Exp Eye Res 2009;89(6):1021-1027. 8.Nien CJ, Massei S, Lin G, et al. Effects of age and dysfunction on human meibomian glands. Arch Ophthalmol 2011;129(4):462-469. 9.Suhalim JL, Parfitt GJ, Xie Y, et al. Effect of desiccating stress on mouse meibomian gland function. Ocul Surf 2014;12(1):59-68. 10.Tombran-Tink J, Johnson LV. Neuronal differentiation of retinoblastoma cells induced by medium conditioned by human RPE cells. Invest Ophthalmol Vis Sci 1989;30(8):1700-1707. 11.Steele FR, Chader GJ, Johnson LV, Tombran-Tink J. Pigment epithelium-derived factor: neurotrophic activity and identification as a member of the serine protease inhibitor gene family. Proc Natl Acad Sci U S A 1993;90(4):1526-1530. 12.Sagheer U, Gong J, Chung C. Pigment Epithelium-Derived Factor (PEDF) is a Determinant of Stem Cell Fate: Lessons from an Ultra-Rare Disease. J Dev Biol 2015;3(4):112-128. 13.He X, Cheng R, Benyajati S, Ma JX. PEDF and its roles in physiological and pathological conditions: implication in diabetic and hypoxia-induced angiogenic diseases. Clin Sci (Lond) 2015;128(11):805-823. 14.Tombran-Tink J, Mazuruk K, Rodriguez IR, et al. Organization, evolutionary conservation, expression and unusual Alu density of the human gene for pigment epithelium-derived factor, a unique neurotrophic serpin. Mol Vis 1996;2:11. 15.Ho TC, Chiang YP, Chuang CK, et al. PEDF-derived peptide promotes skeletal muscle regeneration through its mitogenic effect on muscle progenitor cells. Am J Physiol Cell Physiol 2015;309(3):C159-168. 16.Ho TC, Chen SL, Wu JY, et al. PEDF promotes self-renewal of limbal stem cell and accelerates corneal epithelial wound healing. Stem Cells 2013;31(9):1775-1784. 17.Yeh SI, Ho TC, Chen SL, et al. Pigment Epithelial-Derived Factor Peptide Facilitates the Regeneration of a Functional Limbus in Rabbit Partial Limbal Deficiency. Invest Ophthalmol Vis Sci 2015;56(4):2126-2134. 18.Yeh SI, Ho TC, Chen SL, et al. Pigment Epithelial-Derived Factor Peptide Regenerated Limbus Serves as Regeneration Source for Limbal Regeneration in Rabbit Limbal Deficiency. Invest Ophthalmol Vis Sci 2016;57(6):2629-2636. 19.Lin Z, Liu X, Zhou T, et al. A mouse dry eye model induced by topical administration of benzalkonium chloride. Mol Vis 2011;17:257-264. 20.Wang YC, Li S, Chen X, et al. Meibomian Gland Absence Related Dry Eye in Ectodysplasin A Mutant Mice. Am J Pathol 2016;186(1):32-42. 21.Pult H, Nichols JJ. A review of meibography. Optom Vis Sci 2012;89(5):E760-769. 22.Ho TC, Chen SL, Shih SC, et al. Pigment epithelium-derived factor is an intrinsic antifibrosis factor targeting hepatic stellate cells. Am J Pathol 2010;177(4):1798-1811. 23. Sarojini H, Estrada R, Lu H, et al. PEDF from mouse mesenchymal stem cell secretome attracts fibroblasts. J Cell Biochem 2008;104(5):1793-1802. 24.Li F, Song N, Tombran-Tink J, Niyibizi C. Pigment epithelium-derived factor enhances differentiation and mineral deposition of human mesenchymal stem cells. Stem Cells 2013;31(12):2714-2723. 25.Gattu AK, Swenson ES, Iwakiri Y, et al. Determination of mesenchymal stem cell fate by pigment epithelium-derived factor (PEDF) results in increased adiposity and reduced bone mineral content. FASEB J 2013;27(11):4384-4394. 26.Steinle JJ, Sharma S, Chin VC. Normal aging involves altered expression of growth factors in the rat choroid. J Gerontol A Biol Sci Med Sci 2008;63(2):135-140. 27.Francis MK, Appel S, Meyer C, Balin SJ, Balin AK, Cristofalo VJ. Loss of EPC-1 / PEDF expression during skin aging in vivo. J Invest Dermatol 2004;122(5):1096-1105. 28.Morris DL, Rui L. Recent advances in understanding leptin signaling and leptin resistance. Am J Physiol Endocrinol Metab 2009;297(6):E1247-1259. 29.Knauer DJ, Wiley HS, Cunningham DD. Relationship between epidermal growth factor receptor occupancy and mitogenic response. Quantitative analysis using a steady state model system. J Biol Chem 1984;259(9):5623-5631. 30.Hong I, Lee MH, Na TY, Zouboulis CC, Lee MO. LXRalpha enhances lipid synthesis in SZ95 sebocytes. J Invest Dermatol 2008;128(5):1266-1272. 31.Hwang HS, Parfitt GJ, Brown DJ, Jester JV. Meibocyte differentiation and renewal: Insights into novel mechanisms of meibomian gland dysfunction (MGD). Exp Eye Res 2017;163:37-45. 32.Ho TC, Chen SL, Yang YC, et al. Cytosolic phospholipase A2-{alpha} is an early apoptotic activator in PEDF-induced endothelial cell apoptosis. Am J Physiol Cell Physiol 2009;296(2):C273-284. 33.Ho TC, Chen SL, Yang YC, Liao CL, Cheng HC, Tsao YP. PEDF induces p53-mediated apoptosis through PPAR gamma signaling in human umbilical vein endothelial cells. Cardiovasc Res 2007;76(2):213-223. 34.Liu Y, Zhu Y, Rannou F, et al. Laminar flow activates peroxisome proliferator-activated receptor-gamma in vascular endothelial cells. Circulation 2004;110(9):1128-1133. 35.Notari L, Baladron V, Aroca-Aguilar JD, et al. Identification of a lipase-linked cell membrane receptor for pigment epithelium-derived factor. J Biol Chem 2006;281(49):38022-38037. 36.Yang SL, Chen SL, Wu JY, Ho TC, Tsao YP. Pigment epithelium-derived factor induces interleukin-10 expression in human macrophages by induction of PPAR gamma. Life Sci 2010;87(1-2):26-35. 37.Solomon A, Dursun D, Liu Z, Xie Y, Macri A, Pflugfelder SC. Pro- and anti-inflammatory forms of interleukin-1 in the tear fluid and conjunctiva of patients with dry-eye disease. Invest Ophthalmol Vis Sci 2001;42(10):2283-2292. 38.Tosato G, Jones KD. Interleukin-1 induces interleukin-6 production in peripheral blood monocytes. Blood 1990;75(6):1305-1310. 39.Oliveira IC, Sciavolino PJ, Lee TH, Vilcek J. Downregulation of interleukin 8 gene expression in human fibroblasts: unique mechanism of transcriptional inhibition by interferon. Proc Natl Acad Sci U S A 1992;89(19):9049-9053. 40.Gattu AK, Birkenfeld AL, Iwakiri Y, et al. Pigment epithelium-derived factor (PEDF) suppresses IL-1beta-mediated c-Jun N-terminal kinase (JNK) activation to improve hepatocyte insulin signaling. Endocrinology 2014;155(4):1373-1385. 41.Cavaliere RM, Ghirardi F, Tirindelli R. Lacrimal gland removal impairs sexual behavior in mice. Front Neuroanat 2014;8:101.

Claims

[Claim 1] A pharmaceutical composition comprising PEDF-derived short-chain peptide (PDSP) for use in promoting meibomian gland regeneration or treating and / or preventing dry eye caused by meibomian gland dysfunction, The PDSP is a pharmaceutical composition comprising the sequence SLGAEQRTESIIHRALYYDLISSPDIHGT (Sequence ID 1).

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