Compositions and methods for treating meibomian gland dysfunction

Abstract

The present disclosure provides compositions for use in treating eye-related disorders, such as disorders affecting the meibomian glands. Compositions for use in such disorders may act on the IGF-1 pathway, such as by binding (e.g., agonizing) IGF1R. Other compositions may act on the IGF-1 pathway through other mechanisms. Related methods, kits, and pharmaceutical compositions for treating eye-related disorders are also disclosed.

Description

[Technical Field]

[0001] cross reference This application claims the benefit of U.S. Provisional Application No. 63 / 476,808, filed December 22, 2022, which is incorporated herein by reference in its entirety.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in XML file format and is incorporated herein by reference in its entirety. The original of said XML was created on December 21, 2023, is named 61766-715_601_SL.xml, and is 12,698 bytes in size. [Background technology]

[0003] Meibomian glands are sebaceous glands located primarily along the margins of the upper and lower eyelids. These glands play an important role in preventing ocular dryness by secreting meibum, a mixture of lipids and proteins that coats the surface of the eye and prevents the aqueous component of tears from evaporating. Dysfunction and morphological changes in meibomian glands can lead to dry eye disease (DED). There are no approved medications for treating meibomian gland dysfunction (MGD). While medications exist to address a few symptoms, there are currently no disease-modifying therapies. Approved drugs target inflammation or induce poor-quality aqueous tear production, but they do not provide any long-term improvement to patients. Furthermore, all of these treatments require frequent dosing and high patient compliance to achieve any benefit. Summary of the Invention

[0004] In one aspect, the present disclosure provides a pharmaceutical composition comprising a therapeutically effective amount of an IGF-1 variant that has reduced affinity for at least one IGF-binding protein (IGFBP) compared to the affinity for the interaction between wild-type IGF-1 (SEQ ID NO: 1) and the IGF-binding protein (IGFBP), the pharmaceutical composition being formulated for topical administration. In some embodiments, the pharmaceutical composition is formulated for topical administration to the eye or eyelid.

[0005] In another aspect, the disclosure provides a pharmaceutical composition comprising a therapeutically effective amount of an IGF-1 variant, wherein topical administration of the pharmaceutical composition to the eye or eyelid results in one or more of the following: increased size of the meibomian glands, reduced meibomian gland atrophy, reversal of age-related meibomian gland atrophy, increased function of one or more meibocytes, increased corneal epithelial cell proliferation, increased corneal healing rate, increased IGF1 receptor (IGF1R) activation in the meibomian glands, increased duration of IGF1R activation in the meibomian glands, and increased lipid content of the meibomian glands.

[0006] In some embodiments, the affinity of the IGF-1 variant for at least one IGF-binding protein is reduced compared to the affinity of wild-type IGF-1 for an IGFBP. In some embodiments, the IGF-1 variant has at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to SEQ ID NO: 1. In some embodiments, the pharmaceutical composition is formulated for delivery by eye drops. In some embodiments, the pharmaceutical composition comprises a cream for administration to one or both eyelids of a subject. In some embodiments, the pharmaceutical composition, when administered to a subject suffering from meibomian gland dysfunction, results in an increase in the median surface area or volume of the meibomian glands within the inner eyelid surface of the subject compared to a subject not receiving the pharmaceutical composition. In some embodiments, the pharmaceutical composition, when administered to a subject suffering from meibomian gland dysfunction, results in an increase in the median lipid content within the meibomian glands of the subject compared to a subject not receiving the pharmaceutical composition. In some embodiments, when the pharmaceutical composition is administered to a subject suffering from meibomian gland dysfunction, it results in an increase in the median lipid quality in the subject's meibomian glands compared to a subject not receiving the pharmaceutical composition. In some embodiments, when the pharmaceutical composition is administered to a subject suffering from meibomian gland dysfunction, it results in an increase in the median lipid release from the acini in the subject's meibomian glands compared to a subject not receiving the pharmaceutical composition. In some embodiments, when the pharmaceutical composition is administered to a subject suffering from meibomian gland dysfunction, it results in an increase in the median duration of Akt phosphorylation in meibocytes compared to a subject not receiving the pharmaceutical composition. In some embodiments, the IGF-1 variant is truncated. In some embodiments, the IGF-1 variant comprises or consists of the amino acid sequence of SEQ ID NO: 3. In some embodiments, the IGF-1 variant comprises one or more amino acid substitutions relative to wild-type IGF-1 (SEQ ID NO: 1).In some embodiments, the IGF-1 variant comprises an amino acid deletion at position 37 relative to wild-type IGF-1 (SEQ ID NO: 1), where the numbering of the positional numbers is based on an alignment of the IGF-1 variant with SEQ ID NO: 1, starting at the N-terminus at position 1 and numbering the positions from the N-terminus of SEQ ID NO: 1 to the C-terminus of SEQ ID NO: 1. In some embodiments, the IGF-1 variant comprises the amino acid sequence of SEQ ID NO: 6. In some embodiments, the IGF-1 variant comprises an amino acid substitution at position 60 relative to wild-type IGF-1 (SEQ ID NO: 1), where the numbering of the positional numbers is based on an alignment of the IGF-1 variant with SEQ ID NO: 1, starting at the N-terminus at position 1 and numbering the positions from the N-terminus of SEQ ID NO: 1 to the C-terminus of SEQ ID NO: 1. In some embodiments, the IGF-1 variant comprises the amino acid sequence of SEQ ID NO: 2. In some embodiments, the IGF-1 variant comprises an amino acid substitution at position 3 relative to wild-type IGF-1 (SEQ ID NO: 1), where the numbering of the positional positions is based on an alignment of the IGF-1 variant with SEQ ID NO: 1, starting at position 1 at the N-terminus, and numbering the positions from the N-terminus of SEQ ID NO: 1 to the C-terminus of SEQ ID NO: 1. In some embodiments, the IGF-1 variant comprises the amino sequence of SEQ ID NO: 4. In some embodiments, the IGF-1 variant comprises the amino sequence of SEQ ID NO: 5. In some embodiments, the IGF-1 variant comprises the amino sequence of SEQ ID NO: 7. In some embodiments, the IGF-1 variant comprises the amino sequence of SEQ ID NO: 12. In some embodiments, the IGF-1 variant binds to a cell-penetrating peptide (CPP) or a skin-penetrating peptide (SPP). In some embodiments, the IGF-1 variant binds to a cell-penetrating peptide selected from the group consisting of SEQ ID NO: 10 and SEQ ID NO: 11. In some embodiments, the IGF-1 variant binds to a skin-penetrating peptide of SEQ ID NO: 9. In some embodiments, the IGF-1 variant comprises the amino sequence of SEQ ID NO: 8. In some embodiments, the pharmaceutical composition comprises one or more pharmaceutically acceptable excipients.In some embodiments, the one or more pharmaceutically acceptable excipients comprise one or more of water, saline, sucrose, lactose, malic acid, cellulose sugars, mannitol, maltitol, dextran, sorbitol, starch, agar, alginate, chitin, chitosan, pectin, gum tragacanth, gum arabic, gelatin, collagen, casein, albumin, synthetic or semi-synthetic polymers or glycerides, methylcellulose, hydroxypropylmethylcellulose, and polyvinylpyrrolidone. In some embodiments, the at least one IGFBP comprises IGFBP2. In some embodiments, the at least one IGFBP comprises IGFBP3. In some embodiments, the at least one IGFBP comprises IGFBP1. In some embodiments, the at least one IGFBP comprises IGFBP4. In some embodiments, the at least one IGFBP comprises IGFBP5. In some embodiments, the at least one IGFBP comprises IGFBP6. In some embodiments, administration of the pharmaceutical composition to the subject's eye or eyelid results in an increase in the size of the meibomian gland. In some embodiments, administering the pharmaceutical composition to a subject's eye or eyelid results in a reduction in meibomian gland atrophy. In some embodiments, administering the pharmaceutical composition to a subject's eye or eyelid results in the reversal of age-related meibomian gland atrophy. In some embodiments, administering the pharmaceutical composition to a subject's eye or eyelid results in an increase in the function of one or more meibocytes. In some embodiments, administering the pharmaceutical composition to a subject's eye or eyelid results in an increase in corneal epithelial cell proliferation. In some embodiments, administering the pharmaceutical composition to a subject's eye or eyelid results in an increase in corneal healing rate. In some embodiments, administering the pharmaceutical composition to a subject's eye or eyelid results in an increase in IGF1 receptor (IGF1R) activation in the meibomian glands. In some embodiments, administering the pharmaceutical composition to a subject's eye or eyelid results in an increase in the duration of IGF1R activation in the meibomian glands. In some embodiments, administering the pharmaceutical composition to a subject's eye or eyelid results in an increase in the lipid content of the meibomian glands. In some embodiments, the pharmaceutical composition, when administered to spheroids of IHGMGE cells, results in an increase in the lipid content in the spheroids on average.

[0007] In another aspect, the present disclosure provides a kit comprising a pharmaceutical composition provided herein and an eye dropper for delivering the pharmaceutical composition as an eye drop.

[0008] In another aspect, the present disclosure provides a method for treating an ocular disorder in a subject in need thereof, comprising administering to the subject having the ocular disorder a pharmaceutical composition, wherein the pharmaceutical composition comprises a therapeutically effective amount of an IGF-1 variant having reduced affinity for an IGF-binding protein (IGFBP) compared to the affinity for the interaction between wild-type IGF-1 (SEQ ID NO: 1) and the IGF-binding protein (IGFBP). In some embodiments, the pharmaceutical composition is a pharmaceutical composition provided herein. The pharmaceutical composition is administered to the subject's eye or eyelid. In some embodiments, the pharmaceutical composition is administered to the subject's eye via an eyedropper. In some embodiments, the pharmaceutical composition is administered to the subject's outer eyelid. In some embodiments, the pharmaceutical composition is a cream. In some embodiments, the pharmaceutical composition is administered to a subject suffering from meibomian gland dysfunction. In some embodiments, administering the pharmaceutical composition to the subject results in an increase in the surface area or volume of the meibomian glands within the subject's inner eyelid surface. In some embodiments, administering the pharmaceutical composition to the subject results in an increase in lipid content within the subject's meibomian glands. In some embodiments, administering the pharmaceutical composition to a subject results in increased lipid release from the acini of the meibomian glands of the subject. In some embodiments, administering the pharmaceutical composition to a subject results in an increase in the duration of Akt phosphorylation in meibocytes. In some embodiments, the method does not include administration of any additional phospholipidosis-inducing agent. In some embodiments, the method does not include administration of one or both of azithromycin and doxycycline. In some embodiments, the ocular disorder includes dry eye disease. In some embodiments, the ocular disorder includes meibomian gland dysfunction. The ocular disorder includes Sjogren's syndrome.

[0009] In another aspect, the present disclosure provides a pharmaceutical composition comprising a therapeutically effective amount of a polypeptide comprising the amino acid sequence of any one of SEQ ID NOS: 1-8 or 12 and one or more pharmaceutically acceptable excipients. In some embodiments, the pharmaceutical composition is a solution for delivery as eye drops. In some embodiments, the pharmaceutical composition comprises a cream for administration to one or both eyelids of a subject. In some embodiments, the pharmaceutical composition is formulated for systemic delivery. In some embodiments, the polypeptide is a human IGF1R agonist. In some embodiments, administering the pharmaceutical composition to a subject suffering from meibomian gland dysfunction results in an increase in the median surface area or volume of the meibomian glands within the inner eyelid surface. In some embodiments, administering the pharmaceutical composition to a subject suffering from meibomian gland dysfunction results in an increase in the median lipid content within the meibomian glands. In some embodiments, administering the pharmaceutical composition to a subject suffering from meibomian gland dysfunction results in an increase in the median lipid release from the acini of the meibomian glands. In some embodiments, administering the pharmaceutical composition to a subject suffering from meibomian gland dysfunction results in an increase in median lipid release from the acini of the meibomian glands. In some embodiments, administering the pharmaceutical composition to a subject suffering from meibomian gland dysfunction results in an increase in median Akt phosphorylation in meibocytes. In some embodiments, the pharmaceutical composition does not contain any additional phospholipidosis-inducing agent. In some embodiments, the pharmaceutical composition does not contain any one of azithromycin or doxycycline. In some embodiments, the one or more pharmaceutically acceptable excipients include one or more of water, saline, sucrose, lactose, malic acid, cellulose sugar, mannitol, maltitol, dextran, sorbitol, starch, agar, alginate, chitin, chitosan, pectin, gum tragacanth, gum arabic, gelatin, collagen, casein, albumin, synthetic or semi-synthetic polymers or glycerides, methylcellulose, hydroxypropylmethylcellulose, and polyvinylpyrrolidone. In some embodiments, the polypeptide comprises the amino sequence of SEQ ID NO: 1. In some embodiments, the polypeptide comprises the amino sequence of SEQ ID NO:2.In some embodiments, the polypeptide comprises the amino sequence of SEQ ID NO: 3. In some embodiments, the polypeptide comprises the amino sequence of SEQ ID NO: 4. In some embodiments, the polypeptide comprises the amino sequence of SEQ ID NO: 5. In some embodiments, the polypeptide comprises the amino sequence of SEQ ID NO: 6. In some embodiments, the polypeptide comprises the amino sequence of SEQ ID NO: 7. In some embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NO: 8. In some embodiments, the polypeptide comprises the amino sequence of SEQ ID NO: 12. In some embodiments, the polypeptide further comprises a cell-penetrating peptide (CPP) or a skin-penetrating peptide (SPP). In some embodiments, the polypeptide comprises a cell-penetrating peptide selected from the group consisting of SEQ ID NO: 10 and SEQ ID NO: 11. In some embodiments, the polypeptide comprises the skin-penetrating peptide of SEQ ID NO: 9.

[0010] In another aspect, the present disclosure provides a method for treating an ocular disorder in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising any one of SEQ ID NOS: 1-8 or 12. In some embodiments, the pharmaceutical composition is a pharmaceutical composition provided herein. In some embodiments, the pharmaceutical composition is administered to the eye of the subject. In some embodiments, the pharmaceutical composition is administered to the eye of the subject via an eyedropper. In some embodiments, the pharmaceutical composition is administered to the outer eyelid of the subject. In some embodiments, the pharmaceutical composition is a cream. In some embodiments, administering the pharmaceutical composition to a subject increases the median surface area or volume of the meibomian glands on the inner eyelid surface. In some embodiments, administering the pharmaceutical composition to a subject results in an increase in lipid content in the meibomian glands. In some embodiments, administering the pharmaceutical composition to a subject suffering from meibomian gland dysfunction results in an increase in lipid release from the acini of the meibomian glands. In some embodiments, administering the pharmaceutical composition to a subject results in an increase in lipid release from the acini of the meibomian glands. In some embodiments, administering the pharmaceutical composition to a subject results in an increase in phosphorylation of Akt in meibocytes. In some embodiments, the method does not include administration of any additional phospholipidosis-inducing agent. In some embodiments, the method does not include administration of any one of azithromycin or doxycycline. In some embodiments, the ocular disorder comprises dry eye disease. In some embodiments, the ocular disorder comprises meibomian gland dysfunction. In some embodiments, the ocular disorder comprises Sjogren's syndrome.

[0011]

[0013] Further aspects and advantages of the present disclosure will become readily apparent to those skilled in the art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the present disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.

[0012] Incorporation by Reference All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent that the publications and patents or patent applications incorporated by reference conflict with the disclosure contained herein, the present specification is intended to supersede and / or replace such conflicting material. [Brief explanation of the drawings]

[0013] The novel features of the invention are set forth with particularity in the appended claims. The features and advantages of the present invention will be better understood by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings, in which: [Figure 1] 1 shows the dose-dependent effects of IGF-1 (SEQ ID NO: 1), IGF-1 Ea (SEQ ID NO: 7), IGF-1 Des1-3 R37X (SEQ ID NO: 6), IGF-1 Des1-3 (SEQ ID NO: 8), and IGF-1 E3R (SEQ ID NO: 4) on AKT S473 phosphorylation in DU145 cells. Wild-type IGF-1 and IGF-1 variants have similar EC50 values ​​in DU145 cells. [Figure 2]Figure 1 shows the reduction of IGFBP affinity for various IGF-1 mutants. In a competition assay in the conditioned medium of L6 rat myoblasts containing multiple IGF-binding proteins, it was reported that the BP affinity for IGF-1 LR3 (SEQ ID NO: 12), LG3 (long IGF-1 E3G), long IGF-1, and IGF-1 Des1-3 (SEQ ID NO: 3) was reduced by 690-fold, 112-fold, 5.5-fold, and 38-fold, respectively (see Francis, GL et al., 8(3) J. Mol. Endocrinol. 213-223, 1992). In another study using bovine IGFBP2, it was reported that IGF-1 E3R (SEQ ID NO: 4) and IGF E3G reduced the binding affinity for IGFBP by 230-fold and 59-fold, respectively (see King, R. et al., 8 J. Mol. Endocrinol. 29-41, 1992). [Figure 3A] 3A shows that IGF-1 LR3 (SEQ ID NO: 12) and IGF-1 E3R (SEQ ID NO: 4) are less inhibited by IGFBP2 and IGFBP3 than wild-type IGF-1 (SEQ ID NO: 1). Figure 3A shows the effects of wild-type IGF-1, IGF-1 LR3, and IGF-1 E3R on AKT S473 phosphorylation in DU145 cells in the presence of IGFBP2 at IGF-1 to IGFBP2 ratios of 1:1, 1:2, or 1:4, respectively. [Figure 3B] Figure 3B shows that IGF-1 LR3 (SEQ ID NO: 12) and IGF-1 E3R (SEQ ID NO: 4) are less inhibited by IGFBP2 and IGFBP3 than wild-type IGF-1 (SEQ ID NO: 1). Figure 3B shows the effects of wild-type IGF-1, IGF-1 LR3, and IGF-1 E3R on AKT S473 phosphorylation in DU145 cells in the presence of IGFBP3 at IGF-1 to IGFBP3 ratios of 1:1, 1:2, or 1:4, respectively. [Figure 4]Figure 1 shows the dose-dependent effect of IGF-1 on AKT S473 phosphorylation in immortalized human meibomian gland epithelial cells (IHMGEC). The EC50 value of IGF-1 is approximately 0.07 nanomolar (nM). [Figure 5] The results of a live cell imaging assay are shown, showing that IHMGECs spread and proliferate faster in response to IGF-1 stimulation in a dose-dependent manner. [Figure 6] 1 shows the results of human IGF-1 stimulation of IGHMECs after a cell viability assay, in which the cells proliferate in a dose-dependent manner in response to IGF-1. [Figure 7A] Figure 7 shows that immortalized human meibomian gland epithelial (IHMGE) cells form spheroids resembling meibomian acini when grown in 3D culture. Figure 7A shows images of 2D and 3D spheroid cultures. Brightfield images were collected using transmitted light. [Figure 7B] Figure 7B shows that immortalized human meibomian gland epithelial (IHMGE) cells form spheroids resembling meibomian acini when grown in 3D culture. Immunofluorescence images of spheroids expressing a nuclear marker, a marker for the basal compartment of the meibomian acinar (Krt5), and a merged image of the two are shown. [Figure 8] Figure 1 shows that spheroids grown in the presence of IGF-1 E3R are significantly larger than those grown in wild-type IGF-1. The diameters of spheroids cultured in the presence of 0 nM, 0.1 nM, 1.6 nM, or 10 nM IGF-1 or IGF-1 E3R were measured on day 7 of differentiation. [Figure 9] Figure 1 shows that IGF-1 and IGF-1 E3R proportionally increase lipid content in relation to spheroid diameter. The lipidotox intensity of spheroids cultured in the presence of 0 nM, 0.1 nM, 1.6 nM, or 10 nM IGF-1 or IGF-1 E3R was measured on day 7 of differentiation. [Figure 10]FIG. 1 shows that IGFBP2 was more highly expressed compared to IGFBP1, IGFBP3, IGFBP4, and IGFBP6 in IHMGE spheroid cultures. [Figure 11A] FIG. 1 shows the study design for transcriptional analysis of the effect of wild-type IGF1 treatment on IHMGE spheroids. [Figure 11B] FIG. 1 shows that treatment of IHMGE spheroids with wild-type IGF-1 significantly upregulated four IGFBPs in a transcriptional analysis. [Figure 11C] FIG. 1 shows that IGF-1 treatment significantly upregulates genes involved in fatty acid transport, lipid synthesis, and meibomian gland formation. [Figure 12] Figure 12 shows that IGFBP2 is highly expressed in the basal compartment of mouse meibomian glands. Figure 12 shows representative immunofluorescence images of meibomian glands labeling both nuclei (DAPI, green) and proliferating cells (Ki67, red), IGFBP2, and a merged image of the two. [Figure 13A] Figure 13A shows increased proliferation in meibomian acini of young mice compared to old mice. Cell proliferation in the meibomian glands decreases with age. Figure 13A shows that the number of proliferating cells (Ki67+) in the acini of old meibomian glands was lower than that observed in young meibomian glands. Statistical comparisons were performed using Student's t-test, n = 10 per group. [Figure 13B] Figure 13B shows increased proliferation in meibomian acini of young mice compared to old mice. Cell proliferation in the meibomian gland decreases with age. Figure 13B shows representative immunofluorescence images of young (top) and old (bottom) meibomian glands, labeling nuclei (DAPI, green) and proliferating cells (Ki67, red). Cells positive for both markers in the acini (double positives) are highlighted in blue using image analysis in ImageJ. [Figure 14A]Graphs and images showing increased proliferation in meibomian acini of aged mice with systemic IGF-1 LR3 treatment. Figure 14A shows that IGF-1 LR3 can induce proliferation in the meibomian glands of aged mice. Mice were administered 10 mg / kg of IGF-1 LR3 by intraperitoneal injection twice, 12 hours apart, and sacrificed 24 or 48 hours after the first dose. The number of proliferating cells (Ki67+) in the acini of vehicle-treated (left) or IGF-1 LR3-treated (right) meibomian glands was quantified by comparing the number of Ki67-positive cells in the meibomian acini. Statistical comparisons were performed using two-way ANOVA followed by Tukey's multiple comparison test. N = 10 per group. [Figure 14B] Graphs and images showing increased proliferation in meibomian acini of aged mice with systemic IGF-1 LR3 treatment. Figure 14B shows representative immunofluorescence images of meibomian glands treated with vehicle for 24 hours (top) and IGF-1 LR3 for 24 hours (bottom), labeling nuclei (DAPI, green) and proliferating cells (Ki67, red). Cells positive for both markers in the acini (double positives) are highlighted in blue using image analysis in ImageJ. [Figure 15A] Figure 15A shows graphs and images showing the reversal of atrophy and an increase in meibomian gland area size in aged mice with systemic IGF-1 LR3 treatment. Figure 15A shows mice either untreated (young) or administered 10 mg / kg IGF-1 LR3 by intraperitoneal injection for 5 consecutive days per week for 4 weeks and then subjected to transillumination meibography before sacrifice. Quantification of meibography data shows an increase in gland area in IGF-1 LR3-treated aged mice compared with vehicle-treated control aged mice. Pairwise comparisons were performed using a one-sided Mann-Whitney test, with n = 7–10 mice per group. [Figure 15B]Figure 15B shows graphs and images showing that systemic IGF-1 LR3 treatment reverses atrophy and increases the size of meibomian gland area in aged mice. Figure 15B shows representative images from a transillumination meibography experiment, with the area of ​​one acinus in each image. [Figure 16A] Figure 16A shows graphs and images demonstrating increased lipid synthesis in meibomian glands with systemic IGF-1 LR3 treatment, regardless of age. Figure 16A shows mice either untreated (young) or administered 10 mg / kg IGF-1 LR3 by intraperitoneal injection for 5 consecutive days per week for 4 weeks before sacrifice. Nuclei (DAPI, red) and meibomian gland lipids (Lipidtox, green) were labeled in eyelid sections, and lipid droplet density was quantified using ImageJ. Statistical comparisons were performed using one-way ANOVA followed by Tukey's multiple comparison test. N = 7–10 per group. [Figure 16B] 16A and 16B are graphs and images showing that systemic IGF-1 LR3 treatment increases lipid synthesis in meibomian glands regardless of age. Figure 16B shows representative images from each group. [Figure 17] 1 shows a graph showing that IGF-1 LR3 can be delivered systemically or via eye drops to activate IGF1 receptors in the eyelid. Mice were administered a single dose of 10 mg / kg IGF-1 LR3 via intraperitoneal injection, or treated with vehicle (PBS) or PBS containing 5 mg / ml IGF-1 LR3 via eye drops administered to the ocular surface. 30 minutes after administration, animals were sacrificed, and their eyelids were homogenized and assayed for pAKT levels by pAKT ELISA as a reporter of IGF1R activation. Statistical comparisons were performed using one-way ANOVA followed by Tukey's multiple comparison test. n = 3 per group. [Figure 18]Figure 1 shows that IGF-1 LR3 prolongs the duration of increased IGF1R signaling compared to wild-type IGF-1. The effects of wild-type IGF-1 and IGF-1 LR3 on AKT S473 phosphorylation were measured 0.5 or 2 hours after dosing. Statistical analysis: One-way Anova. n = 3 per group. [Figure 19]

[0039] Figure 1 shows that both IGF-1 LR3 and IGF-1 Des 1-3, which have reduced binding capacity to binding proteins, prolong the duration of IGF1R activation 2 hours post-dosing compared to wild-type IGF-1. n=5 / group. The effects of IGF-1, IGF-1 LR3, and IGF-1 Des 1-3 on AKT S473 phosphorylation were measured in the eyelids of tested animals 2 hours post-dosing. Statistical analysis: One-way Anova. [Figure 20] Figure 1 shows that IGF-1 LR3 and IGF-1 E3R, which have reduced binding capacity to binding proteins, have similar in vivo efficacy compared to IGF-1 at 1 hour post-dose. The effects of IGF-1, IGF-1 LR3, and IGF-1 E3R on AKT S473 phosphorylation were measured in the eyelids of tested animals at 1 hour post-dose. n=3 / group. Statistical analysis: One-way Anova. [Figure 21] Figure 1 shows that IGF-1 LR3 stimulates dose-responsive proliferation in the meibomian glands of aged mice. The number of Ki67-labeled proliferating cells per 100 μm circumference was measured in young or aged mice after treatment with 0 mg / ml, 0.3 mg / ml, 1 mg / ml, or 3 mg / ml IGF-1 LR3 eye drops. n=5 / group. Statistical analysis: One-way Anova. [Figure 22A] FIG. 1 shows that IGF-1 LR3 regenerates atrophied meibomian glands in aged mice. [Figure 22B]Figure 22 shows that IGF-1 LR3 regenerates atrophied meibomian glands in aged mice. Daily treatment with IGF-1 LR3 for one month increases meibomian gland area, comparing pre- and post-treatment. Figure 22A shows the glands of individual animals before and after IGF-1 LR3 or vehicle treatment. Figure 22B shows quantification of the change in gland area with IGF-1 LR3 or vehicle treatment, as assessed by transillumination meibography. n=10 / group. Statistical analysis: One-way Anova. [Figure 23] Figure 1 shows that IGF-1 LR3 and IGF-1 E3R induce similar levels of proliferation in the meibomian glands of aged mice after 2 weeks of daily eye drop administration. The number of Ki67-labeled proliferating cells per 100 μm circumference was measured in young or aged mice after treatment with vehicle or IGF-1 ER3 eye drops. n=4-5 / group. Statistical analysis: One-way Anova. DETAILED DESCRIPTION OF THE INVENTION

[0014] The present disclosure should not be limited to the specific embodiments described in this application, which are intended as examples of individual aspects of the disclosure. Not all various embodiments of the present disclosure will be described herein. As will be apparent to those skilled in the art, many modifications and variations are possible without departing from the spirit and scope of the present disclosure. Functionally equivalent methods and apparatuses within the scope of the present disclosure, in addition to those enumerated herein, will be apparent to those skilled in the art from the foregoing description. Such modifications and variations are intended to be included within the scope of the appended claims. The present disclosure is limited only by the language of the appended claims, along with the full scope of equivalents to which such claims are entitled.

[0015] Specific Definitions All terms are intended to be understood as understood by one of ordinary skill in the art. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0016] The following definitions supplement those in the art and are directed to the present application. These definitions do not apply to any related or unrelated case, for example, any commonly owned patents or applications. Although any methods and materials similar or equivalent to those described herein can be used in carrying out the tests of the present disclosure, the preferred materials and methods are described herein. Therefore, the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0017] The terms used herein are for the purpose of describing particular cases only and are not intended to be limiting. In this application, the use of the singular includes the plural unless specifically stated otherwise. As used herein, the singular forms "a," "an," and "the" are intended to include the plural as well unless the context clearly indicates otherwise.

[0018] In this application, the use of "or" means "and / or" unless otherwise specified. The terms "and / or" and "any combinations thereof," and their grammatical equivalents used herein, may be used interchangeably. These terms may convey that any and all combinations are specifically contemplated. For illustrative purposes only, the phrase "A, B, and / or C" or "A, B, C, or any combinations thereof" may mean "A individually, B individually, C individually, A and B, B and C, A and C, and A, B, and C." The term "or" may be used consecutively or disjunctively unless the context specifically dictates disjunctive use.

[0019] The term "about" or "approximately" means plus or minus 10% of the number to which the term refers.

[0020] As used in the specification and claims, the words "comprising" (and any form of comprising, such as "comprise" or "comprises"), "having" (and any form of having, such as "have" or "has"), "including" (and any form of including, such as "includes" or "include"), or "containing" (and any form of containing, such as "contains" or "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or steps. Any embodiment discussed in this specification can be implemented with respect to any method or composition of the disclosure, and vice versa. Furthermore, the compositions of the disclosure can be used to achieve the methods of the disclosure.

[0021] References herein to "some embodiments," "an embodiment," "one embodiment," or "other embodiments" mean that the particular features, structures, or characteristics described in connection with those embodiments are included in at least some embodiments of the disclosure, but not necessarily in all embodiments. To facilitate understanding of this disclosure, a number of terms and phrases are defined below.

[0022] Ranges provided herein are understood to be abbreviations for all values ​​within that range. For example, the range 1 to 50 is understood to include any number, combination of numbers, or subrange from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50, and further includes all decimal points between the recited integers, such as, for example, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, and 1.9. With respect to subranges, "nested sub-ranges" extending from either end of the range are specifically contemplated. For example, nested sub-ranges of the exemplary range of 1 to 50 could include 1 to 10, 1 to 20, 1 to 30, and 1 to 40 in one direction, and 50 to 40, 50 to 30, 50 to 20, and 50 to 10 in another direction.

[0023] The term "pharmaceutically acceptable" means approved or approvable by a federal or state regulatory agency, or listed in the United States Pharmacopeia (USP) or other generally recognized pharmacopeia, for use in animals, including humans.

[0024] A "pharmaceutically acceptable excipient, carrier, or diluent" refers to an excipient, carrier, or diluent that can be administered to a subject together with a drug, does not destroy its pharmacological activity, and is non-toxic when administered in an amount sufficient to deliver a therapeutic amount of the drug.

[0025] The term "subject" refers to an animal that is the object of treatment, observation, or experiment. By way of example only, a subject includes, but is not limited to, a human or non-human mammal, e.g., a mammal, including, but not limited to, a non-human primate such as a cow, horse, dog, sheep, or cat.

[0026] The term "optional" or "optionally" indicates that the event or circumstance described below may occur, but need not occur, and that the description includes both cases where the event or circumstance occurs and cases where it does not. The term "sequence identity" or percent (%) sequence identity, as used herein, refers to the percentage of residues in a candidate sequence that are identical to those in a selected sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity; any conservative substitutions are not considered as part of the sequence identity. Alignment to determine percent amino acid sequence identity can be achieved in a variety of ways within the skill of those in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms necessary to achieve maximum alignment across the entire length of the sequences being compared.

[0027] The term "effective amount" or "therapeutically effective amount" refers to an amount of an agent sufficient to produce a beneficial or desired outcome. The therapeutically effective amount may vary depending on one or more of the subject and disease state being treated, the subject's weight and age, the severity of the disease state, the method of administration, etc., which can be easily determined by one of ordinary skill in the art. The term "effective amount" also applies to a dose that provides an image for detection by an appropriate imaging method. A particular dose may vary depending on one or more of the particular agent selected, the dosing regimen to be followed, whether it is administered in combination with other compounds, the timing of administration, the tissue to be imaged, the physical delivery system that carries it, etc. An effective amount of an active agent may be administered in a single dose or multiple doses.

[0028] The terms "polypeptide," "oligopeptide," "peptide," and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. The polymers may be linear or branched, may contain modified amino acids, and may be interrupted by non-amino acids. These terms also encompass amino acid polymers that are modified naturally or by intervention, for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. The definition also includes, for example, polypeptides containing one or more amino acid analogs (including, for example, unnatural amino acids), as well as other modifications known in the art.

[0029] While specific embodiments of the present application have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, modifications, and substitutions may occur to those skilled in the art without departing from these embodiments. It should be understood that various alternatives to the embodiments described herein may be employed in carrying out the methods described herein.

[0030] Polypeptides Meibomian glands contain meibocytes, which are cells that form clusters in acini. Under normal function, meibocytes can proliferate, differentiate, and produce meibum, a lipid-rich secretion. Dysfunction of the meibomian glands can be a catalyst in the development of dry eye disease. In one exemplary mechanism, reduced binding of insulin-like growth factor (IGF-1) to IGF-1 receptor (IGF1R), resulting in reduced IGF1R activation (or other IGF1R signal transduction defects), can affect the morphology and function of the meibomian glands. For example, this can lead to meibomian gland atrophy and dysfunction, or dry eye disease.

[0031] The present disclosure provides polypeptides and treatment methods for meibomian gland or dry eye disease. The polypeptides can modulate (e.g., upregulate or otherwise activate) the activity of IGF1R. The polypeptides may have reduced affinity for at least one IGF-binding protein (IGFBP) compared to wild-type IGF-1 (SEQ ID NO: 1) for the IGFBP. In one embodiment, the polypeptides act locally. The levels of one or both of (1) free endogenous IGF-1 or (2) polypeptides (IGF-1 variants with reduced affinity for one or more IGFBPs) that bind to IGF1R can be increased upon administration to a subject. Local activation of IGF1R by the polypeptides can result in prolonged or extended pharmacodynamic effects. In another embodiment, the polypeptides act systemically. The systemic half-life of the polypeptides is reduced due to their reduced affinity for IGFBPs.

[0032] IGF-1 and IGF-1 variants can bind to one or more IGFBPs. IGFBPs can extend the half-life of circulating wild-type IGF-1. In one aspect, IGFBPs can act to enhance systemic IGF-1 signaling by increasing the levels of circulating IGF-1 and IGF-1 variants. In another aspect, IGFBPs can act to attenuate local IGF-1 signaling by decreasing the levels of local free IGF-1 and IGF-1 variants. In one aspect, the one or more IGFBPs may include one or more of IGFBP1, IGFBP2, IGFBP3, IGFBP4, IGFBP5, IGFBP6, or any combination thereof. In some embodiments, the one or more IGFBPs include IGFBP1. In some embodiments, the one or more IGFBPs include IGFBP2. In some embodiments, the one or more IGFBPs include IGFBP3. In some embodiments, the one or more IGFBPs include IGFBP4. In some embodiments, the one or more IGFBPs include IGFBP1 and IGFBP2. In some embodiments, the one or more IGFBPs include IGFBP1 and IGFBP3. In some embodiments, the one or more IGFBPs include IGFBP1 and IGFBP4. In some embodiments, the one or more IGFBPs include IGFBP2 and IGFBP3. In some embodiments, the one or more IGFBPs include IGFBP2 and IGFBP3. In some embodiments, the one or more IGFBPs include IGFBP3 and IGFBP4. In some embodiments, the one or more IGFBPs include IGFBP1, IGFBP2, and IGFBP3. In some embodiments, the one or more IGFBPs include IGFBP1, IGFBP2, and IGFBP4. In some embodiments, the one or more IGFBPs include IGFBP1, IGFBP3, and IGFBP4. In some embodiments, the one or more IGFBPs include IGFBP2, IGFBP3, and IGFBP4. In some embodiments, the one or more IGFBPs include IGFBP1, IGFBP2, IGFBP3, and IGFBP4.

[0033] [Table 1]

[0034] In some embodiments, the polypeptide contains or consists of SEQ ID NO: 1 (native human IGF-1). The polypeptide may contain or consist of 70 amino acids. The polypeptide may have strong binding affinity to IGF1R. The polypeptide may have strong binding affinity to one or more IGFBPs. The affinity of native IGF-1 for IGF1R may be in the range of about 1 nanomolar (nM) to about 10 nM, as measured by affinity assays.

[0035] In one aspect, the polypeptide is an IGF-1 variant. In some embodiments, the IGF-1 variant has at least 60% sequence identity to SEQ ID NO: 1. In some embodiments, the IGF-1 variant has at least 65% sequence identity to SEQ ID NO: 1. In some embodiments, the IGF-1 variant has at least 70% sequence identity to SEQ ID NO: 1. In some embodiments, the IGF-1 variant has at least 75% sequence identity to SEQ ID NO: 1. In some embodiments, the IGF-1 variant has at least 80% sequence identity to SEQ ID NO: 1. In some embodiments, the IGF-1 variant has at least 85% sequence identity to SEQ ID NO: 1. In some embodiments, the IGF-1 variant has at least 90% sequence identity to SEQ ID NO: 1. In some embodiments, the IGF-1 variant has at least 91% sequence identity to SEQ ID NO: 1. In some embodiments, the IGF-1 variant has at least 92% sequence identity to SEQ ID NO: 1. In some embodiments, the IGF-1 variant has at least 93% sequence identity to SEQ ID NO: 1. In some embodiments, the IGF-1 variant has at least 94% sequence identity to SEQ ID NO: 1. In some embodiments, the IGF-1 variant has at least 95% sequence identity to SEQ ID NO: 1. In some embodiments, the IGF-1 variant has at least 96% sequence identity to SEQ ID NO: 1. In some embodiments, the IGF-1 variant has at least 97% sequence identity to SEQ ID NO: 1. In some embodiments, the IGF-1 variant has at least 98% sequence identity to SEQ ID NO: 1. In some embodiments, the affinity of the IGF-1 variant for IGF1R may range from about 1 nanomolar (nM) to about 10 nM, as measured by an affinity assay. In some embodiments, the polypeptide (e.g., the IGF-1 variant) has an EC50 of greater than 1 nM, greater than 2 nM, greater than 3 nM, greater than 4 nM, or greater than 5 nM, as determined by the assay described in connection with FIG. 1.In some embodiments, the EC50 of the polypeptide is between 1 nM and 20 nM, between 1 nM and 15 nM, between 1 nM and 10 nM, between 2 nM and 10 nM, or between 3 nM and 5 nM. In some embodiments, the polypeptide (e.g., an IGF-1 variant) does not bind to the insulin receptor, or binds very weakly. For example, in some cases, the Kd of the interaction of the polypeptide (e.g., an IGF-1 variant) is more than 10-fold weaker than the binding of insulin to the insulin receptor.

[0036] In some embodiments, the IGF-1 variant comprises a truncation of the amino acid sequence of SEQ ID NO: 1. In some embodiments, the truncation is at the N-terminus of SEQ ID NO: 1. In some embodiments, the truncation is at the C-terminus of SEQ ID NO: 1. In some embodiments, the IGF-1 variant comprises a truncation comprising a deletion of at least about 1 amino acid. In some embodiments, the IGF-1 variant comprises a truncation comprising a deletion of at least about 2 amino acids. In some embodiments, the IGF-1 variant comprises a truncation comprising a deletion of at least about 3 amino acids. In some embodiments, the IGF-1 variant comprises a truncation comprising a deletion of at least about 4 amino acids. In some embodiments, the IGF-1 variant comprises a truncation comprising a deletion of at least about 5 amino acids. In some embodiments, the IGF-1 variant comprises a truncation comprising a deletion of at least about 6 amino acids. In some embodiments, the IGF-1 variant comprises a truncation comprising a deletion of at least about 7 amino acids. In some embodiments, the IGF-1 variant comprises a truncation comprising a deletion of at least about 8 amino acids. In some embodiments, the IGF-1 variant comprises a truncation comprising a deletion of at least about 9 amino acids. In some embodiments, the IGF-1 variant comprises a truncation comprising a deletion of at least about 10 amino acids. In some embodiments, the IGF-1 variant comprises a truncation comprising a deletion of at least about 11 amino acids. In some embodiments, the IGF-1 variant comprises a truncation comprising a deletion of at least about 12 amino acids.

[0037] In some embodiments, the IGF-1 variant is an extension of the amino acid sequence of SEQ ID NO: 1. In some embodiments, the extension is at the C-terminus of SEQ ID NO: 1. In some embodiments, the extension is at the N-terminus of SEQ ID NO: 1. In some embodiments, the extension has at least about 2 amino acids. In some embodiments, the extension has at least about 3 amino acids. In some embodiments, the extension has at least about 4 amino acids. In some embodiments, the extension has at least about 5 amino acids. In some embodiments, the extension has at least about 6 amino acids. In some embodiments, the extension has at least about 7 amino acids. In some embodiments, the extension has at least about 8 amino acids. In some embodiments, the extension has at least about 9 amino acids. In some embodiments, the extension has at least about 10 amino acids. In some embodiments, the extension has at least about 15 amino acids. In some embodiments, the extension has at least about 20 amino acids. In some embodiments, the extension has at least about 35 amino acids. In some embodiments, the extension has at least about 40 amino acids. In some embodiments, the extension has at least about 45 amino acids. In some embodiments, the extension has at least about 50 amino acids.

[0038] In some embodiments, the IGF-1 variant comprises one or more amino acid substitutions relative to wild-type IGF-1 (SEQ ID NO: 1). In some embodiments, the IGF-1 variant comprises an amino acid substitution at position 3 relative to wild-type IGF-1 (SEQ ID NO: 1). In some embodiments, the amino acid substitution at position 3 is arginine. In some embodiments, the IGF-1 variant comprises an amino acid substitution at position 60 relative to wild-type IGF-1 (SEQ ID NO: 1). In some embodiments, the amino acid substitution at position 60 is leucine. The numbering of positions can be based on an alignment of the IGF-1 variant with SEQ ID NO: 1, with the N-terminus of SEQ ID NO: 1 being numbered as position 1, from the N-terminus of SEQ ID NO: 1 to the C-terminus of SEQ ID NO: 1. In some embodiments, the polypeptide comprises IGF-1 Y60L. In some embodiments, the polypeptide comprises IGF-1 E3R. In some embodiments, the polypeptide comprises native IGF-1 with a deletion at R37.

[0039] In some embodiments, the IGF-1 variant comprises a truncation of the amino acid sequence of SEQ ID NO: 1 and one or more amino acid substitutions relative to wild-type IGF-1 (SEQ ID NO: 1). In some embodiments, the IGF-1 variant comprises a truncation of three amino acids at the N-terminus of SEQ ID NO: 1 and an amino acid deletion at position 37 relative to wild-type IGF-1 (SEQ ID NO: 1). In some embodiments, the IGF-1 variant comprises a deletion relative to SEQ ID NO: 1. In some embodiments, the IGF-1 variant comprises an amino acid deletion at position 37 relative to wild-type IGF-1 (SEQ ID NO: 1).

[0040] In some embodiments, the IGF-1 variant comprises the amino sequence of SEQ ID NO: 2. In some embodiments, the IGF-1 variant comprises the amino sequence of SEQ ID NO: 3. In some embodiments, the IGF-1 variant comprises the amino sequence of SEQ ID NO: 4. In some embodiments, the IGF-1 variant comprises the amino sequence of SEQ ID NO: 5. In some embodiments, the IGF-1 variant comprises the amino sequence of SEQ ID NO: 6. In some embodiments, the IGF-1 variant comprises the amino sequence of SEQ ID NO: 7. In some embodiments, the IGF-1 variant comprises the amino sequence of SEQ ID NO: 8. In some embodiments, the IGF-1 variant comprises the amino sequence of SEQ ID NO: 12.

[0041] The polypeptide may comprise or consist of about 66 amino acids. The polypeptide may comprise or consist of about 67 amino acids. The polypeptide may comprise or consist of about 69 amino acids. The polypeptide may comprise or consist of about 150 amino acids. The polypeptide may comprise or consist of about 70 amino acids. The polypeptide may comprise or consist of about 83 amino acids. The polypeptide may comprise or consist of about 105 amino acids.

[0042] The polypeptide may have a reduced systemic and / or local half-life when administered to a healthy adult compared to native human IGF-1. In some embodiments, when administered to a healthy adult, the systemic and / or local half-life of the polypeptide is reduced by at least 5% compared to native human IGF-1. In some embodiments, when administered to a healthy adult, the systemic and / or local half-life of the polypeptide is reduced by at least 10% compared to native human IGF-1. In some embodiments, when administered to a healthy adult, the systemic and / or local half-life of the polypeptide is reduced by at least 15% compared to native human IGF-1. In some embodiments, when administered to a healthy adult, the systemic and / or local half-life of the polypeptide is reduced by at least 20% compared to native human IGF-1. In some embodiments, when administered to a healthy adult, the systemic and / or local half-life of the polypeptide is reduced by at least 30% compared to native human IGF-1. In some embodiments, when administered to a healthy adult, the systemic and / or local half-life of the polypeptide is reduced by at least 40% compared to native human IGF-1. In some embodiments, when administered to a healthy adult, the systemic and / or local half-life of the polypeptide is reduced by at least 50% compared to native human IGF-1.

[0043] The polypeptide may have reduced affinity for at least one IGFBP compared to native human IGF-1 (SEQ ID NO: 1) for that IGFBP. In some embodiments, the polypeptide has reduced affinity for IGFBP1 compared to native human IGF-1 (SEQ ID NO: 1). In some embodiments, the polypeptide has reduced affinity for IGFBP2 compared to native human IGF-1 (SEQ ID NO: 1). In some embodiments, the polypeptide has reduced affinity for IGFBP3 compared to native human IGF-1 (SEQ ID NO: 1). In some embodiments, the polypeptide has reduced affinity for IGFBP4 compared to native human IGF-1 (SEQ ID NO: 1). In some embodiments, the polypeptide has reduced affinity for IGFBP5 compared to native human IGF-1 (SEQ ID NO: 1). In some embodiments, the polypeptide has reduced affinity for IGFBP6 compared to native human IGF-1 (SEQ ID NO: 1). In some embodiments, the polypeptide has reduced affinity for IGFBP3 and IGFBP2 compared to native human IGF-1 (SEQ ID NO: 1).

[0044] In some embodiments, the polypeptide may further comprise a 13 amino acid sequence (MFPAMPLLSLFVN (SEQ ID NO: 13)) at its C-terminal or N-terminal sequence.

[0045] IGFBPs play a key role in extending the systemic half-life of insulin-like growth factor 1 (IGF-1) in humans. While the unbound form of wild-type IGF-1 has a systemic half-life of 10–20 minutes, binding of IGF-1 to IGFBPs extends its half-life to several hours. This extended half-life is generally thought to extend the duration of IGF-1's biological effects. This is most clearly seen in Laron syndrome, a type of dwarfism in which patients are unable to produce IGFBPs specifically. Laron dwarfs treated with high doses of systemic IGF1 fail to thrive, in part because IGF-1 cannot persist in the circulation without the dramatic half-life extension provided by IGFBP binding.

[0046] Therefore, as described herein, it is surprising that when an IGF-1 variant that avoids IGFBPs is administered topically (e.g., as an eye drop to treat meibomian gland dysfunction), the opposite phenomenon is observed. For example, the desired pharmacodynamic effects of wild-type IGF-1 treatment are observed at lower levels and over a shorter time frame than IGF-1 variants (e.g., IGF1 LR3, IGF des 1-3, IGF E3R) that have the ability to avoid one or more IGFBPs. In other words, although it is understood that binding to one or more binding proteins is important for extending the half-life and function of IGF-1, unexpectedly, compared to wild-type IGF-1 (SEQ ID NO: 1), variants that avoid one or more IGF-1 binding proteins (e.g., IGFBP2) can exhibit extended and / or improved pharmacodynamic effects when administered topically.

[0047] Cell-penetrating peptides (CPPs) and skin-penetrating peptides (SPPs) The polypeptides disclosed herein may further comprise a cell-penetrating peptide (CPP) or a skin-penetrating peptide (SPP). The stratum corneum of the skin generally comprises keratin-rich dead cells suspended in layered lipid domains, which act as a barrier to the environment. This structure can inhibit the absorption and transport of macromolecules into the dermis and beyond. The CPPs or SPPs disclosed herein can have high transduction efficiency, thereby enabling transdermal delivery. The CPPs or SPPs disclosed herein can modify the structure of the skin barrier, allowing molecules co-formulated with them (even if not conjugated) to penetrate and / or translocate across the skin.

[0048] The IGF-1 variant can be conjugated to a cell-penetrating peptide (CPP) or skin-penetrating peptide (SPP) described herein. The CPP or SPP may comprise any one of SEQ ID NOs: 9-11. In some embodiments, the CPP or SPP comprises SEQ ID NO: 9. In some embodiments, the CPP or SPP comprises SEQ ID NO: 10. In some embodiments, the CPP or SPP comprises SEQ ID NO: 11. In some embodiments, a polypeptide comprising SEQ ID NO: 1-8, 12, or any combination thereof binds to a CPP or SPP. In some embodiments, a polypeptide comprising SEQ ID NO: 1 binds to a CPP or SPP. In some embodiments, a polypeptide comprising SEQ ID NO: 2 binds to a CPP or SPP. In some embodiments, a polypeptide comprising SEQ ID NO: 3 binds to a CPP or SPP. In some embodiments, a polypeptide comprising SEQ ID NO: 4 binds to a CPP or SPP. In some embodiments, a polypeptide comprising SEQ ID NO: 5 binds to a CPP or SPP. In some embodiments, a polypeptide comprising SEQ ID NO: 6 binds to a CPP or SPP. In some embodiments, a polypeptide comprising SEQ ID NO: 7 binds to a CPP or SPP. In some embodiments, a polypeptide comprising SEQ ID NO: 8 binds to a CPP or SPP. In some embodiments, a polypeptide comprising SEQ ID NO: 12 binds to a CPP or SPP.

[0049] A composition or formulation delivered transdermally may be configured to achieve a steady-state level better than a composition or formulation not delivered transdermally. A composition or formulation delivered transdermally may bypass liver metabolism and reduce unwanted systemic side effects, thereby improving patient compliance. The CPPs or SPPs disclosed herein may comprise or consist of about 5 to about 30 amino acids. The CPPs or SPPs disclosed herein may enable the polypeptide to penetrate the skin of the eyelid, thereby allowing the polypeptide to be absorbed into the eyelid. The SPPs disclosed herein may comprise or consist of a hydrophobic peptide. The CPPs disclosed herein may comprise or consist of a cationic peptide. The cationic peptide may have one or more charged amino acids, such as arginine. The CPPs or SPPs may exhibit low cytotoxicity to cells, e.g., human cells. The SPPs disclosed herein may comprise a macromolecular transduction domain (MTD). The MTD comprises or consists of MRAAAPAVAA (SEQ ID NO: 9). The MTD may be derived from the membrane-transmitting sequence (MTS) of the Kaposi's fibroblast growth factor (FGF-4) signal peptide. The CPP disclosed herein comprises or consists of SEQ ID NO: 10. The polypeptide of SEQ ID NO: 10 can penetrate the epidermis and dermis of the skin and may be useful for treating antioxidant disorders. The polypeptide of SEQ ID NO: 10 may be delivered, for example, via a skin spray. The CPP disclosed herein comprises or consists of SEQ ID NO: 11. The polypeptide of SEQ ID NO: 11 can penetrate the epidermis and dermis of the skin and may be useful for treating antioxidant disorders. The polypeptide of SEQ ID NO: 11 may be delivered, for example, via a skin spray.

[0050] Pharmaceutical Composition The present disclosure provides a pharmaceutical composition for treating meibomian gland or dry eye disease. The pharmaceutical composition may be applied topically, for example, as eye drops or as a cream. The pharmaceutical composition may be applied to the eyelid. The pharmaceutical composition may be formulated for systemic delivery. The pharmaceutical composition may penetrate the eyelid.

[0051] The pharmaceutical composition may include a pharmaceutically acceptable carrier or adjuvant, such as, for example, hyaluronate (or hyaluronic acid), electrolytes, ophthalmic demulcents, excipients, astringents, vasoconstrictors, and / or emollients. Examples of pharmaceutically acceptable excipients may include one or more pharmaceutically acceptable excipients including one or more of water, saline, sucrose, lactose, malic acid, cellulose sugars, mannitol, maltitol, dextran, sorbitol, starch, agar, alginate, chitin, chitosan, pectin, tragacanth gum, arabic gum, gelatin, collagen, casein, albumin, synthetic or semi-synthetic polymers or glycerides, methylcellulose, hydroxypropylmethylcellulose, and polyvinylpyrrolidone. Examples of electrolytes may include sodium chloride, potassium chloride, sodium bicarbonate, potassium bicarbonate, calcium chloride, magnesium chloride, trisodium citrate, hydrochloric acid, sodium hydroxide, and mixtures thereof. The pharmaceutical compositions disclosed herein may include a solution having one or more electrolytes. For example, in some embodiments, the electrolyte-containing solution includes one or more of sodium chloride, potassium chloride, sodium bicarbonate, potassium bicarbonate, calcium chloride, magnesium chloride, trisodium citrate, hydrochloric acid, or sodium hydroxide. In some examples, the molar percentage of sodium chloride is about 40% to about 60%. In some examples, the molar percentage of sodium chloride is about 40%, 45%, 50%, 55%, or 60%. In some examples, the molar percentage of potassium chloride is about 1% to about 20%. In some examples, the molar percentage of potassium chloride is about 1%, 2%, 3%, 4%, 5%, 10%, 15%, or 20%. In some examples, the molar percentage of sodium bicarbonate is about 1% to about 25%. In some examples, the mole percent of sodium bicarbonate is about 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, or 25%. In some examples, the mole percent of potassium bicarbonate is about 0% to about 10%. In some examples, the mole percent of potassium bicarbonate is about 0%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%.In some examples, the molar percentage of calcium chloride is about 0% to 10%. In some examples, the molar percentage of calcium chloride is about 0%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%. In some examples, the molar percentage of magnesium chloride is about 0% to 10%. In some examples, the molar percentage of magnesium chloride is about 0%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%. In some examples, the molar percentage of trisodium citrate is about 0% to 10%. In some examples, the molar percentage of trisodium citrate is about 0%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%. In some examples, the molar percentage of hydrochloric acid is about 0% to about 30%. In some examples, the molar percentage of hydrochloric acid is about 0%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, or 30%. In some examples, the molar percentage of sodium hydroxide is about 0% to about 30%. In some examples, the molar percentage of sodium hydroxide is about 0%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, or 30%.

[0052] In some embodiments, the excipient may include one or more penetration enhancers. The term "permeation enhancer" or "penetration enhancer" may refer to a compound or mixture of compounds that interacts with one or more skin components to enhance drug penetration into and / or through the skin (e.g., through the skin of the outer eyelid to one or more meibomian glands). Any suitable penetration enhancer known in the art can be used in the pharmaceutical compositions described herein, including, for example, surfactants (e.g., ionic (anionic, cationic, zwitterionic) surfactants (e.g., sodium lauryl sulfate, sodium laurate, etc.), non-ionic surfactants (e.g., Tween-80, other polysorbates, etc.), and any combination thereof), bile salts and derivatives thereof (e.g., sodium glycolate, sodium deoxycholate, etc.), fatty acids and derivatives thereof (e.g., fatty acid esters of oleic acid, caprylic acid, isopropyl myristate, etc.), chelating agents (e.g., EDTA, citric acid, etc.), sulfoxides (e.g., DMSO, DMA, DMF, etc.), polyols (e.g., diethylene glycol monoesters, ethyl ether, PG, polyethylene glycol (PEG), glycerol, polyglycols, etc.), alcohols (e.g., alkanols, alkenols, glycols, etc.), hydrocarbons (e.g., alkanes, alkenes, halogenated alkanes, squalene, squalene, mineral oils, etc.), amines, amides (e.g., cyclic amides, acyclic amides, azones, pyrrolidones, urea and its derivatives, etc.), others (e.g., terpenes and terpenoids, essential oils (e.g., eucalyptus oil, peppermint oil, turpentine, etc.), phospholipids, cyclic oligosaccharides (e.g., cyclodextrins), amino acids and thioacyl derivatives of amino acids, alkylamino esters and oxazolidinones, enzymes, ketones (e.g., macrocyclic ketones), etc.), hyaluronic acid, benzalkonium chloride, and any combination thereof.

[0053] In some embodiments, the pharmaceutically acceptable excipient is adapted for transdermal delivery through the outer eyelid by formulating the excipient to 1) achieve enhanced spreadability on the outer eyelid surface and / or 2) prevent flow from the outer eyelid surface to the corneal surface. In some embodiments, the pharmaceutically acceptable excipient comprises properties and rheological characteristics that enhance spreadability, resulting in easier administration and spreading on the eyelid surface and a lack of flowability at a subject's body temperature, particularly after application to the subject's skin. In some embodiments, the pharmaceutical compositions of the present disclosure are formulated so that the cohesiveness of the formulation does not significantly change after application to a subject's skin (e.g., eyelid). Examples of suitable additives that provide suitable cohesiveness of the formulation include additives that increase the viscosity of the formulation, such as waxes, paraffins, and elastomers. In some embodiments, the viscosity of the formulation does not significantly change when heated from room temperature to a temperature closer to the subject's body temperature.

[0054] In some embodiments, the pharmaceutically acceptable formulation is an ointment comprising a water-miscible ointment base. In some embodiments, the pharmaceutically acceptable formulation is an ointment comprising a paraffin-based ointment base. In some embodiments, the ointment comprises one or more of white petrolatum, mineral oil, propylene glycol, ST cyclomethicone 5NF, Labrasol, propylene carbonate, steareth 2, ST emulsifier 10, and ST elastomer 10. In some embodiments, the ointment comprises white petrolatum, mineral oil, propylene glycol, ST cyclomethicone 5NF, Labrasol, propylene carbonate, steareth 2, ST emulsifier 10, and ST elastomer 10.

[0055] In some embodiments, the pharmaceutically acceptable formulation is a cream comprising an oil-in-water base. In some embodiments, the pharmaceutically acceptable formulation is a cream comprising a water-in-oil base. In some embodiments, the cream comprises one or more of white petrolatum / petrolatum, mineral oil, propylene glycol, cyclomethicone, ST cyclomethicone 5NF, emulsifier 10, ST emulsifier, ST elastomer 10, methylparaben, disodium hydrogen phosphate, citric acid, propylparaben, and purified water. In some embodiments, the cream comprises white petrolatum / petrolatum, mineral oil, propylene glycol, ST cyclomethicone 5NF, ST emulsifier, ST elastomer 10, methylparaben, disodium hydrogen phosphate, citric acid, propylparaben, and purified water. In some embodiments, the cream comprises white petrolatum / petrolatum, mineral oil, propylene glycol, cyclomethicone, emulsifier 10, ST elastomer 10, methylparaben, disodium hydrogen phosphate anhydrous, citric acid anhydrous, propylparaben, and purified water.

[0056] The pharmaceutical composition disclosed herein may be administered to a subject. A therapeutically effective amount of the pharmaceutical composition disclosed herein may be administered to a subject. The pharmaceutical composition may be administered by any delivery mode disclosed herein, for example, eye drops or cream. Eye drops may be administered via an eye dropper. The pharmaceutical composition may be administered to the subject's eye or a portion thereof. The pharmaceutical composition may be administered to the eyelid, for example, the outer eyelid.

[0057] The subject may suffer from meibomian gland dysfunction.Meibomian gland dysfunction can lead to changes in tear film composition, ocular surface disease, eye and eyelid discomfort, and evaporative dry eye.Symptoms of meibomian gland dysfunction include dryness, burning sensation, itching, redness, crusty secretions, tearing, blurred vision, and light sensitivity.

[0058] In some embodiments, administering a pharmaceutical composition disclosed herein to a subject may result in an increase in meibomian gland size compared to a subject not administered the pharmaceutical composition. In some embodiments, administering a pharmaceutical composition disclosed herein to a subject may result in a decrease in meibomian gland atrophy compared to a subject not administered the pharmaceutical composition. In some embodiments, administering a pharmaceutical composition disclosed herein to a subject may result in meibomian gland regeneration compared to a subject not administered the pharmaceutical composition. In some embodiments, administering a pharmaceutical composition disclosed herein to a subject may result in the reversal of age-related meibomian gland atrophy compared to a subject not administered the pharmaceutical composition. In some embodiments, administering a pharmaceutical composition disclosed herein to a subject may result in an increase in the function of one or more meibocytes compared to a subject not administered the pharmaceutical composition. For example, in some instances, an increase in function may include an increase in phosphorylation of Akt (or an increase in the duration of Akt activity), an increase in IGF1R itself, or an increase in other downstream signaling molecules that are phosphorylated when IGF1R is activated. In some embodiments, administering a pharmaceutical composition disclosed herein to a subject may result in increased corneal proliferation and / or repair compared to a subject not administered the pharmaceutical composition. In some embodiments, administering a pharmaceutical composition disclosed herein to a subject may result in increased corneal healing compared to a subject not administered the pharmaceutical composition. In some embodiments, administering a pharmaceutical composition disclosed herein to a subject may result in increased IGF1 receptor (IGF1R) activation in the meibomian glands compared to a subject not administered the pharmaceutical composition. In some embodiments, administering a pharmaceutical composition disclosed herein to a subject may result in an increased duration of IGF1R activation in the meibomian glands compared to a subject not administered the pharmaceutical composition. In some embodiments, administering a pharmaceutical composition disclosed herein to a subject may result in an increased lipid content of the meibomian glands compared to a subject not administered the pharmaceutical composition.

[0059] Administration of a pharmaceutical composition to a subject suffering from meibomian gland dysfunction may result in an increase in the median surface area or volume of the subject's meibomian glands compared to a subject not receiving the pharmaceutical composition. In some embodiments, the increase in surface area or volume of the meibomian glands within the subject's inner eyelid surface is at least 5% compared to a subject not receiving the pharmaceutical composition. In some embodiments, the increase in surface area or volume of the meibomian glands in the subject is at least 6% compared to a subject not receiving the pharmaceutical composition. In some embodiments, the increase in surface area or volume of the meibomian glands in the subject is at least 7% compared to a subject not receiving the pharmaceutical composition. In some embodiments, the increase in surface area or volume of the meibomian glands in the subject is at least 8% compared to a subject not receiving the pharmaceutical composition. In some embodiments, the increase in surface area or volume of the meibomian glands in the subject is at least 9% compared to a subject not receiving the pharmaceutical composition. In some embodiments, the increase in surface area or volume of the meibomian glands in the subject is at least 10% compared to a subject not receiving the pharmaceutical composition. In some embodiments, the surface area or volume of the meibomian glands of the subject is increased by at least 15% compared to a subject not receiving the pharmaceutical composition. In some embodiments, the surface area or volume of the meibomian glands of the subject is increased by at least 20% compared to a subject not receiving the pharmaceutical composition. In some embodiments, the surface area or volume of the meibomian glands of the subject is increased by at least 30% compared to a subject not receiving the pharmaceutical composition. In some embodiments, the surface area or volume of the meibomian glands of the subject is increased by at least 40% compared to a subject not receiving the pharmaceutical composition. In some embodiments, the surface area or volume of the meibomian glands of the subject is increased by at least 50% compared to a subject not receiving the pharmaceutical composition. In some embodiments, the surface area or volume of the meibomian glands of the subject is increased by at least 60% compared to a subject not receiving the pharmaceutical composition.

[0060] Administration of a pharmaceutical composition to a subject suffering from meibomian gland dysfunction may result in an increase in median lipid content in the meibomian glands compared to a subject not receiving the pharmaceutical composition. In some embodiments, the increase in lipid content in the meibomian glands is at least 5% compared to a subject not receiving the pharmaceutical composition. In some embodiments, the increase in lipid content in the meibomian glands is at least 6% compared to a subject not receiving the pharmaceutical composition. In some embodiments, the increase in lipid content in the meibomian glands is at least 7% compared to a subject not receiving the pharmaceutical composition. In some embodiments, the increase in lipid content in the meibomian glands is at least 8% compared to a subject not receiving the pharmaceutical composition. In some embodiments, the increase in lipid content in the meibomian glands is at least 9% compared to a subject not receiving the pharmaceutical composition. In some embodiments, the increase in lipid content in the meibomian glands is at least 10% compared to a subject not receiving the pharmaceutical composition. In some embodiments, the increase in lipid content in the meibomian glands is at least 15% compared to a subject not receiving the pharmaceutical composition. In some embodiments, the increase in lipid content in the meibomian glands is at least 20% compared to a subject not receiving the pharmaceutical composition. In some embodiments, the increase in lipid content in the meibomian glands is at least 30% compared to a subject not receiving the pharmaceutical composition. In some embodiments, the increase in lipid content in the meibomian glands is at least 40% compared to a subject not receiving the pharmaceutical composition. In some embodiments, the increase in lipid content in the meibomian glands is at least 50% compared to a subject not receiving the pharmaceutical composition. In some embodiments, the increase in lipid content in the meibomian glands is at least 60% compared to a subject not receiving the pharmaceutical composition.

[0061] Administration of a pharmaceutical composition to a subject suffering from meibomian gland dysfunction may result in an increase in median lipid quality in the meibomian glands compared to subjects not receiving the pharmaceutical composition. Lipid quality may be assessed by having a lower melting point (e.g., less waxy) than lipids (e.g., meibum) from subjects not receiving the pharmaceutical composition. In other words, lipids (e.g., meibum) with a lower average melting point may be of improved quality relative to lipids (e.g., meibum) with a higher average melting point. In some embodiments, the increase in lipid quality in the meibomian glands is at least 5% compared to subjects not receiving the pharmaceutical composition. In some embodiments, the increase in lipid quality in the meibomian glands is at least 6% compared to subjects not receiving the pharmaceutical composition. In some embodiments, the increase in lipid quality in the meibomian glands is at least 7% compared to subjects not receiving the pharmaceutical composition. In some embodiments, the increase in lipid quality in the meibomian glands is at least 8% compared to subjects not receiving the pharmaceutical composition. In some embodiments, the increase in lipid quality in the meibomian glands is at least 9% compared to subjects not receiving the pharmaceutical composition. In some embodiments, the increase in lipid quality in the meibomian glands is at least 10% compared to a subject not receiving the pharmaceutical composition. In some embodiments, the increase in lipid quality in the meibomian glands is at least 15% compared to a subject not receiving the pharmaceutical composition. In some embodiments, the increase in lipid quality in the meibomian glands is at least 20% compared to a subject not receiving the pharmaceutical composition. In some embodiments, the increase in lipid quality in the meibomian glands is at least 30% compared to a subject not receiving the pharmaceutical composition. In some embodiments, the increase in lipid quality in the meibomian glands is at least 40% compared to a subject not receiving the pharmaceutical composition. In some embodiments, the increase in lipid quality in the meibomian glands is at least 50% compared to a subject not receiving the pharmaceutical composition. In some embodiments, the increase in lipid quality in the meibomian glands is at least 60% compared to a subject not receiving the pharmaceutical composition.

[0062] Administration of a pharmaceutical composition to a subject suffering from meibomian gland dysfunction may result in an increase in median lipid release from the acini of the meibomian glands compared to a subject not receiving the pharmaceutical composition. In some embodiments, the increase in lipid release from the acini of the meibomian glands is at least 5% compared to a subject not receiving the pharmaceutical composition. In some embodiments, the increase in lipid release from the acini of the meibomian glands is at least 10% compared to a subject not receiving the pharmaceutical composition. In some embodiments, the increase in lipid release from the acini of the meibomian glands is at least 15% compared to a subject not receiving the pharmaceutical composition. In some embodiments, the increase in lipid release from the acini of the meibomian glands is at least 20% compared to a subject not receiving the pharmaceutical composition. In some embodiments, the increase in lipid release from the acini of the meibomian glands is at least 30% compared to a subject not receiving the pharmaceutical composition. In some embodiments, the increase in lipid release from the acini of the meibomian glands is at least 40% compared to a subject not receiving the pharmaceutical composition. In some embodiments, the increase in lipid release from the acini of the meibomian glands is at least 50% compared to a subject not receiving the pharmaceutical composition. In some embodiments, the increase in lipid release from the acini of the meibomian glands is at least 75% compared to a subject not receiving the pharmaceutical composition. In some embodiments, the increase in lipid release from the acini of the meibomian glands is at least 100% compared to a subject not receiving the pharmaceutical composition. In some embodiments, the increase in lipid release from the acini of the meibomian glands is at least 150% compared to a subject not receiving the pharmaceutical composition. In some embodiments, the increase in lipid release from the acini of the meibomian glands is at least 200% compared to a subject not receiving the pharmaceutical composition. In some embodiments, the increase in lipid release from the acini of the meibomian glands is at least 300% compared to a subject not receiving the pharmaceutical composition.

[0063] Administration of a pharmaceutical composition to a subject suffering from meibomian gland dysfunction may result in an increase in one or more pharmacodynamic effects, such as an increase in the duration of Akt phosphorylation in meibocytes, compared to a subject not administered the pharmaceutical composition. In some embodiments, the increase in the duration of Akt phosphorylation in meibocytes is at least 5% compared to a subject not administered the pharmaceutical composition. In some embodiments, the increase in the duration of Akt phosphorylation in meibocytes is at least 10% compared to a subject not administered the pharmaceutical composition. In some embodiments, the increase in the duration of Akt phosphorylation in meibocytes is at least 15% compared to a subject not administered the pharmaceutical composition. In some embodiments, the increase in the duration of Akt phosphorylation in meibocytes is at least 20% compared to a subject not administered the pharmaceutical composition. In some embodiments, the increase in the duration of Akt phosphorylation in meibocytes is at least 30% compared to a subject not administered the pharmaceutical composition. In some embodiments, the increase in the duration of Akt phosphorylation in meibocytes is at least 40% compared to a subject not administered the pharmaceutical composition. In some embodiments, the increase in the duration of Akt phosphorylation in meibocytes is at least 50% compared to a subject not administered the pharmaceutical composition. In another embodiment, the change in one or more pharmacodynamic effects can be an increase in phosphorylation of any downstream target resulting from activation of IGF1R.

[0064] A pharmaceutical composition may comprise a polypeptide disclosed herein. The polypeptide may comprise the sequence of any one of SEQ ID NOs: 1-8 or 12. The polypeptide may be an IGF-1 variant. The IGF-1 variant may comprise or consist of the sequence of any one of SEQ ID NOs: 2-8 or 12. The pharmaceutical composition may comprise an IGF-1 variant having reduced affinity for at least one IGFBP compared to wild-type IGF-1 (SEQ ID NO: 1).

[0065] In some embodiments, the pharmaceutical composition does not include any additional phospholipidosis-inducing agents. In some embodiments, the pharmaceutical composition does not include either azithromycin or doxycycline.

[0066] Methods of Treatment, Administration, and Use Also provided herein are methods for treating a disease or condition in a subject in need thereof. Also provided herein are methods for treating an ocular disorder in a subject in need thereof. In some aspects, the method comprises administering a pharmaceutical composition provided herein. In some aspects, the method comprises administering a polypeptide provided herein. In some aspects, the method comprises administering an IGF-1 variant provided herein. In some embodiments of the methods disclosed herein, the pharmaceutical composition is administered topically. In some embodiments, the subject is a human. In some embodiments, a therapeutically effective amount of a pharmaceutical composition provided herein is administered.

[0067] Also provided herein is the use of the pharmaceutical compositions provided herein in the treatment of ocular disorders.

[0068] The subject may have a particular disease or condition requiring treatment provided by the present disclosure. The disease or condition may include dry eye disease, meibomian gland dysfunction, and / or Sjogren's syndrome.

[0069] For treatment, the amount of the pharmaceutical composition provided herein administered is an amount effective to produce the desired effect, for example, treatment or alleviation of the effects and / or symptoms of an eye disorder in a subject in need thereof. The effective amount can be provided in a single or series of administrations of the pharmaceutical composition provided herein.

[0070] Subjects suffering from an eye disorder can be identified by any diagnostic or prognostic assay known in the art, or a combination thereof.

[0071] The method for treating a subject in need thereof may further comprise administering to the subject at least one additional therapy, e.g., artificial tears or punctal plugs, either sequentially, separately, or simultaneously.

[0072] In any case, multiple therapeutic agents can be administered in any order, or even simultaneously.If simultaneously, multiple therapeutic agents can be provided in a single integrated form or in multiple forms (for example, as a single tablet or as two separate tablets).One of the therapeutic agents can be administered in multiple doses, or both can be administered in multiple doses.If not simultaneously, the timing between multiple doses can vary from more than 0 weeks to less than 4 weeks.In addition, combination methods, compositions, and preparations are not limited to the use of only two drugs.

[0073] kit The present disclosure provides a kit comprising a pharmaceutical composition disclosed herein and an eye dropper, which may be configured to deliver the pharmaceutical composition as eye drops. [Example]

[0074] Example 1: In vitro potency of wild-type IGF-1 and IGF-1 variants in the presence and absence of IGFBPs

[0075] material and method

[0076] In vitro potency assay in DU145 cells

[0077] 15 x 10 frozen down at passage 5 (P5) 6DU145 cells (ATCC #HTB-81) were replated and grown in a T175 culture flask containing DMEM / F12 (1:1) medium (Gibco 11320-033) supplemented with 10% FBS (Gibco 10437-028) and 10 μg / mL gentamicin (Gibco 15710-064) (hereafter "culture medium"). At 80-90% confluency, cells were trypsinized, manually counted using a hemocytometer, and cultured at 3 x 10 cells / ml in culture medium. 5 The cells were resuspended at 1000 cells / mL and then replated in 0.5 mL aliquots per well (150,000 cells / well) into 24-well plates (Corning 3524). 14–16 hours after replated, the culture medium was removed, washed with 1 mL of PBS, and then cultured in 225 μL of serum-free culture medium supplemented with 0.2% BSA (Millipore #A3059) for 6 hours. 25 μL aliquots of a growth factor dilution series of the following: IGF-1 (SEQ ID NO: 1), IGF-1 Ea (SEQ ID NO: 7), IGF-1 Des1-3 R37X (SEQ ID NO: 6), IGF-1 Des1-3 (SEQ ID NO: 8), and IGF-1 E3R (SEQ ID NO: 4) were added to the cells in duplicate to achieve final concentrations in the wells of 457, 137, 41, 12.5, 3.7, 1.11, and 0.33 nM. After 15 min of incubation at 37°C, the medium was aspirated and cells were lysed in 150 μL of 1x PTR buffer (Extraction Buffer 5x PTR, Abcam ab193970) containing 1x Extraction Enhancer Buffer (Extraction Enhancer Buffer 50x, Abcam ab193971) and protease / phosphatase inhibitors (one Pierce minitablet [#A32959] per 10 mL of 1x PTR). After 15–30 min of incubation on ice, the plates were vortexed, and the lysates were transferred to Eppendorf tubes and stored at −80°C for assessment of IGF1R-AKT signal intensity using the Human / Mouse / Rat Phosphorylated Akt (S473) Pan-Specific DuoSet IC ELISA (RnD #DYC887B).

[0078] Culture of IGFBPs and IGF-1 in DU145 cells

[0079] To measure the in vitro potency of IGF-1 and IGF-1 variants in the presence of IGFBPs, DU145 cells were prepared as described above. During this time, IGFBPs were incubated with 250 nM rhIGF1 (SEQ ID NO: 1), rhIGF1 LR3 (SEQ ID NO: 12), or rhIGF1 E3R (SEQ ID NO: 4) at a molar ratio of 1:1, 2:1, or 4:1. After incubation at room temperature for 1 hour, complexes containing IGF1 variants at a final concentration of 25 nM were added to the cells in triplicate.

[0080] result

[0081] The dose-dependent effects of IGF-1 (SEQ ID NO: 1), IGF-1 Ea (SEQ ID NO: 7), IGF-1 Des1-3 R37X (SEQ ID NO: 6), IGF-1 Des1-3 (SEQ ID NO: 8), and IGF-1 E3R (SEQ ID NO: 4) on AKT S473 phosphorylation were measured in DU145 cells. 50 The EC value of IGF-1 Ea was 6.0 nM. 50 The EC value of IGF-1 Des 1-3 R37X was 8.7 nM. 50 The EC value of IGF-1 Des 1-3 was 4.1 nM. 50 The EC value of IGF-1 E3R was 2.3 nM. 50 The value was 4.5 nM, as shown in Figure 1. This indicates that wild-type IGF-1 and the tested IGF-1 variants have similar ECE in vitro. 50 This suggests that it has value.

[0082] A literature search was conducted to determine differences in the ability of various IGF1 variants to bind to IGFBPs. The results of this search are shown in Figure 2. Competition assays in conditioned medium of L6 rat myoblasts containing multiple binding proteins reported a 690-fold, 112-fold, 5.5-fold, and 38-fold reduction in BP affinity for IGF-1 LR3 (SEQ ID NO: 12), LG3 (long IGF-1 E3G), long IGF-1, and IGF-1 Des1-3 (SEQ ID NO: 3), respectively (see Francis, GL et al., 8(3) J. Mol. Endocrinol. 213-223, 1992). Another study using bovine IGFBP2 reported that IGF-1 E3R (SEQ ID NO: 4) and IGF E3G reduced the binding affinity to IGFBP2 by 230-fold and 59-fold, respectively (see King, R. et al., 8 J. Mol. Endocrinol. 29-41, 1992).This suggests that some IGF-1 variants, such as IGF-1 Des1-3 (SEQ ID NO: 3), IGF-1 E3R (SEQ ID NO: 4), and IGF-1 LR3 (SEQ ID NO: 12), have reduced affinity to IGFBP.

[0083] To measure the efficacy of IGF-1 variants in the presence of IGFBPs, pAKT levels were measured in DU145 cells after preincubation of IGFBP2 or IGFBP3 with wild-type IGF-1 (SEQ ID NO: 1), IGF-1 LR3 (SEQ ID NO: 12), or IGF-1 E3R (SEQ ID NO: 4) at IGF-1 protein to IGFBP ratios of 1:1, 1:2, or 1:4. Figures 3A and 3B suggest that IGF-1 LR3 (SEQ ID NO: 12) and IGF-1 E3R (SEQ ID NO: 4) are less inhibited by IGFBP2 and IGFBP3 than wild-type IGF-1 (SEQ ID NO: 1).

[0084] Example 2: Wild-type IGF-1 promotes spheroid growth.

[0085] material and method

[0086] In vitro potency assay (pAKT assay)

[0087] 5 x 10 frozen at passage 5 (P5) 6 Immortalized human meibomian gland epithelial cells (IHMGEC: ATCC #CRL-3472) were replated and grown in T75 culture flasks containing KSFM medium (Gibco: #10724-011) ("Growth Medium") supplemented with 5 μg / L human recombinant EGF (Gibco #10450-013) and bovine pituitary extract (BPE) (Gibco #13028-014), MEM NEAA (Gibco #11140-050), Pen Strep (Gibco #15140-122). At 90-95% confluency, cells were trypsinized and counted using an EVE automated cell counter from NanoEntek, at a concentration of 4 × 10 cells in culture medium. 5Cells were resuspended at 10000 cells / ml and then replated in 1 ml aliquots per well in 12-well plates (ThermoFisher #FB012928). 36-42 hours after replated, the culture medium was removed, the cells were washed twice with 1 ml of PBS, and then cultured for 6 hours in 450 μl of DMEM / F12 (1:1) medium (Gibco: #11320-033) supplemented with Pen-Strep (Gibco #15140-122) and 0.2% BSA (Millipore #A3059) ("serum starvation medium"). 50 μl aliquots of the following dilution series of rh-IGF-1 (SEQ ID NO: 1) were added to the cells in duplicate: 10000, 1000, 100, 10, 1, 0.1, and 0 nM. After 15 min of incubation at 37°C, the culture medium was aspirated and cells were lysed in 100 μl of M-PER (Thermo Scientific #78501) containing protease / phosphatase inhibitors (one Pierce Minitablet [#A32961] per 10 ml of M-PER). After 15–30 min of incubation on ice, the plates were vortexed, and the lysates were transferred to Eppendorf tubes and stored at −80°C for assessment of IGFR1-AKT signal intensity using the Phosphorylated (S473) Pan Specific DuoSet IC ELISA (RnD #DYC887B-2). ELISA was performed in 384-well plates according to the manufacturer's recommendations with minor modifications. Briefly, wells of a 384-well plate were coated with 25 μl of 6 μg / ml phosphorylated Akt1 (S473) capture antibody (#841692) in PBS (25 μl per 384-well plate), sealed, and incubated overnight at room temperature. The wells were washed four times with 100 μl of PBS containing 0.05% Tween 20 and blocked with 50 μl of PBS containing 2% BSA for 1-2 hours at room temperature. The wells were washed four times with 100 μl of PBS containing 0.05% Tween 20 and incubated with 25 μl of lysate or P-AKT standard for 2 hours at room temperature.Wells were washed four times with 100 μl of PBS containing 0.05% Tween 20 and then incubated with 25 μl of 100 ng / ml phosphorylated Akt1 (S473) detection antibody (#843081) diluted in PBS containing 1% BSA for 1 hour at room temperature. Wells were washed four times with 100 μl of PBS containing 0.05% Tween 20 and then incubated with 25 μl of streptavidin-HRP A (RnD# 890803) diluted 1:200 in PBS containing 1% BSA for 20 minutes at room temperature. Wells were washed four times with 100 μl of PBS containing 0.05% Tween 20 and then incubated with 25 μl of TMB substrate solution (Abcam TMB ELISA Substrate High Sensitivity (ab171523; lot GR3427893-1)) for 10–20 minutes at room temperature. Add 12.5 μl of stop solution (RnD# 895926 from Ancillary Kit 2) and measure the OD of each well using a BMG Labtech CLARIOstar® Plus Microplate Reader. 450 ECE was measured. 50 Values ​​were calculated using Prism 9.

[0088] In vitro human meibomian gland epithelial cell proliferation assay

[0089] Live cell imaging assay. 5 × 10 cryopreserved cells at passage 5 (P5) 6 Immortalized human meibomian gland epithelial cells (IHMGEC: ATCC #CRL-3472) were replated and expanded in a T75 culture flask containing growth medium. At 90-95% confluency, cells were trypsinized, counted using an EVE automated cell counter from NanoEntek, and plated at 1 x 10 in growth medium. 5The cells were resuspended at 1000 cells / ml and then replated in 1 ml aliquots per well into two 12-well plates (ThermoFisher #FB012928). Thirty-six hours after replated, the growth medium was removed, the cells were washed twice with 1 ml of PBS, and then cultured in 500 μl of serum-starvation medium supplemented with 0, 0.1, 1, 10, 100, or 1000 nM human LR3-IGF-1 (PeproTech: #100-11R3) (four independent wells per condition). At 24-hour intervals, cells in the center of each well were imaged using an Eclipse Ti microscope (Nikon) equipped with a 10x Plan Fluor Ph1 lens (Nikon) and an iXon Life 888 EMCCD camera.

[0090] CellTiter-Glo luminescent cell viability assay. 5 x 10 frozen at passage 5 (P5) 6 Immortalized human meibomian gland epithelial cells (IHMGEC: ATCC #CRL-3472) were replated and expanded in a T75 culture flask containing growth medium. At 90-95% confluency, cells were trypsinized, counted using an EVE automated cell counter from NanoEntek, and plated at 1 x 10 in growth medium. 5Cells were resuspended at 1000 cells / ml and then replated in 1 ml aliquots per well into two 12-well plates (ThermoFisher FB012928). Thirty-six hours after replated, the growth medium was removed, the cells were washed twice with 1 ml of PBS, and then cultured in 500 μl of serum-starvation medium supplemented with 0, 0.1, 1, 10, 100, or 1000 nM human IGF-1 (R&D 291-G1). After 58 hours, the medium in each well was replaced with 200 μl of DMEM / F12 and an equal volume of CellTiter-Glo® Reagent. After 10 minutes on an orbital shaker, two 100 μl aliquots from each of the 12 wells were transferred to a 96-well flat-bottom black fluotrac plate (Greiner #655076), and light emission was measured at 560-580 nm using a BMG Labtech CLARIOstar® Plus Microplate Reader. As a measure of IGF-1-mediated proliferation of the cells, the RLU values ​​of IHMGECs treated with IGF-1 were normalized to IHMGECs cultured in the absence of human IGF-1.

[0091] result

[0092] Figure 4 shows the dose-dependent effect of IGF-1 on AKT S473 phosphorylation in IHMGEC. 50 The value is approximately 0.07 nanomolar (nM). Figure 5 shows the results of a live cell imaging assay, in which IHMGECs spread and proliferate faster in response to IGF-1 stimulation in a dose-dependent manner. Figure 6 shows the results of human IGF-1 stimulation in IGHMECs after a cell viability assay, in which the cells proliferate in response to IGF-1 in a dose-dependent manner.

[0093] Example 3: IGF1 E3R promotes spheroid growth more potently than wild-type IGF-1.

[0094] material and method

[0095] 3D culture of IHGMGE cells

[0096] 2500 immortalized human meibomian gland epithelial cells (IHMGEC, ATCC, #CRL-3472) were seeded per well of a 24-well plate in a drop of 50 μl of matrix (Matrigel, Corning #354230) overlaid with growth medium (keratinocyte serum-free medium (Gibco, #17005042) supplemented with 50 μg / mL bovine pituitary extract (Gibco, #13028-014), 5 ng / mL recombinant human epidermal growth factor (EGF, Gibco, #10450-013), 10 μg / mL gentamicin (Gibco, #15710-064), and 1% Pen / Strep (Gibco, #15140-122)). Spheroids were grown in growth medium for 8 days, followed by a 7-day differentiation stage in the presence of differentiation medium (DMEM:F12 medium (Gibco #11320-033) supplemented with or without 10 μg / mL gentamicin (Gibco, #15710-064), 1% Pen / Strep (Gibco, #15140-122), 20 μM rosiglitazone (Sigma Aldrich, #R2408), and varying amounts of recombinant human insulin-like growth factor-1 (rhIGF1, PeproTech #100-11) or IGF1-E3R (SEQ ID NO: 12). N = 2 per condition.

[0097] On day 6 of differentiation, spheroids were fixed with 4% PFA and processed for immunofluorescence using an antibody against Krt5 (Purified anti-Keratin 5 Polyclonal Chicken Antibody, Biolegend #905903) and detected with an AF594-conjugated secondary anti-chicken antibody (Goat anti-Chicken IgY(H+L) Secondary Antibody, Alexa Fluo 594, Thermo #A-11042). For images of 2D and 3D spheroid cultures, brightfield images were collected under transmitted light.

[0098] Measurement of spheroid growth of IHGMGE cells

[0099] IHMGE cells were cultured as described above. As a measure of proliferation, the diameter of each spheroid was measured on day 7 of differentiation using Fiji 2 in ImageJ. To this end, a z-stack of 25 bright-field images spanning a total of 1 mm was taken, and a maximum image projection was used to generate one final image for measurement.

[0100] Measurement of lipid production in spheroids

[0101] IHMGE cells were cultured as described above. On day 6 of differentiation, 1:3000 LipidTOX Green neutral lipid stain (Invitrogen, #H34475) was added to the wells. As a measure of lipid production, on day 7 of differentiation, a z-stack of 25 4x images spanning 1 mm was acquired using a fluorescence microscope with a filter set appropriate for Alexa Fluor 488 dye or fluorescein. After maximum image projection, the average signal per spheroid was calculated using QuPath software (https: / / qupath.github.io / ).

[0102] Quantification of IGFBPs in spheroids

[0103] IHMGE cells were cultured as described above. On day 6 of differentiation, replicates were pooled and lysed in 200 μl of 2x extraction buffer (5X PTR, Abcam #, ab193970) and vortexed thoroughly. Lysates were then frozen at -80°C. For analysis, protein quantification was performed on each sample using the Pierce Detergent Compatible Bradford Assay Kit (Thermo, #23246) and analyzed by Human IGF Signaling Array C1 (Ray Biotech, #AAH-IGF-1-4) according to the manufacturer's instructions.

[0104] result

[0105] IHMGE cells grown in 3D culture under differentiation-promoting conditions developed into spheroid structures resembling meibomian acini, as shown in Figures 7A and 7B. As shown in Figures 7A and 7B, the spheroids expressed markers of the basal compartment of the meibomian acinar, including Krt5, and exhibited proliferation in the basal compartment.

[0106] The perimeter of spheroids cultured in the presence of 0 nM, 0.1 nM, 1.6 nM, or 10 nM IGF-1 or IGF-1 E3R was measured on day 6 of differentiation. Figure 8 shows that spheroids grown in the presence of IGF-1 E3R were significantly larger than those grown in wild-type IGF-1. Furthermore, the Lipidtox intensity of spheroids cultured in the presence of 0 nM, 0.1 nM, 1.6 nM, or 10 nM IGF-1 or IGF-1 E3R was measured on day 7 of differentiation. As shown in Figure 9, IGF-1 and IGF-1 E3R proportionally increase the lipid content in spheroids. Cells were lysed to quantify IGFBPs. Figure 10 shows that IGFBP2 was more highly expressed in IHMGE spheroid cultures compared to IGFBP1, IGFBP3, and IGFBP4.

[0107] Example 4: IGF1 treatment upregulates genes involved in fatty acid transport, lipid synthesis, and meibomian gland formation

[0108] material and method

[0109] 2500 immortalized human meibomian gland epithelial cells (IHMGEC, ATCC, #CRL-3472) were seeded per well of a 24-well plate in a drop of 50 μl of matrix (Geltrex, ThermoFisher #A1413201) overlaid with growth medium (keratinocyte serum-free medium (Gibco, #17005042) supplemented with 50 μg / mL bovine pituitary extract (Gibco, #13028-014), 5 ng / mL recombinant human epidermal growth factor (EGF, Gibco, #10450-013), 10 μg / mL gentamicin (Gibco, #15710-064), and 1% Pen / Strep (Gibco, #15140-122)). Spheroids were grown in growth medium for 14 days, followed by a 4-day differentiation stage in the presence of differentiation medium (DMEM:F12 medium (Gibco #11320-033) supplemented with or without 10 μg / mL gentamicin (Gibco, #15710-064), 1% Pen / Strep (Gibco, #15140-122), 20 μM rosiglitazone (Sigma-Aldrich, #R2408), and 100 nM recombinant human insulin-like growth factor-1 (rhIGF1, PeproTech #100-11)).

[0110] RNA extraction from 4-day differentiated spheroid cultures was performed using TRIzol extraction (Invitrogen #15596026) according to the manufacturer's instructions, followed by an RNA cleanup step using RNA Clean & Concentrator-5 (Zymo Research #R1013). Briefly, n = 4 samples per condition. The matrix droplet containing the spheroids was transferred to an Eppendorf tube, and 500 μL of TRIzol was added to lyse the cells. 100 μL of chloroform was then added, and the aqueous phase was transferred to an RNase-free tube and mixed with an equal volume of 100% ethanol. The mixture was cleaned up using a Zymo-Spin IC column, including DNase I treatment, all according to the protocol. RNA quality and quantity were assessed using Nanodrop before submitting the total RNA samples to Novogene (A260 / A280 = 1.8-2, A260 / A230 >= 1.8). Novogene confirmed the purity and integrity of the RNA. Next, mRNA libraries were prepared using polyA enrichment and subsequently tested for quality (library QC).The libraries were then sequenced on an Illumina NovaSeq PE150 platform using a 150 bp paired-end sequencing strategy (6 G of raw data per sample), and the quality of the resulting data was also confirmed (data QC).

[0111] Fastq files of paired-end RNA-Seq reads were aligned to the reference genome hg38 (Schneider, VA et al., 27(5) Genome research 849-864, 2017) using STAR v.2.6.0a (Dobin, A. et al., 29(1) Bioinformatics 15-21, 2013). Gene-level counts from read pairs were obtained using FeatureCounts v.2.0.6 from the SubRead package (Liao, Y. et al., 30(7) Bioinformatics 923-930, 2014). After removing genes with an average raw count below 10, differential expression analysis was performed using the R package DESeq2 v.1.40.2 (Love, MI et al., 15(12) Genome biology 550, 2014). Gene Set Enrichment Analysis (GSEA) (Subramanian, A. et al., 102(43) Proc Natl Acad Sci U S A. 15545-15550, 2005) was performed to detect enriched pathways for a human gene set from MSigDB (Liberzon, A. et al., 27(12) Bioinformatics 1739-1740, 2011) using a gene list ranked in descending order by log2 fold change. Overrepresentation analysis was performed on a gene set related to meibomian gland formation compiled from literature using Fisher's exact test for selected upregulated (log2 fold change > 1, adjusted p-value < 0.25) and downregulated (log2 fold change < -1, adjusted p-value < 0.25) genes. Heatmaps were generated using Morpheus (Broad Institute).

[0112] result

[0113] To perform transcriptional analysis of the effects of wild-type IGF1 treatment on IHMGE spheroids, IHMGE cells were seeded in Matrigel and differentiated for 4 days in the presence of rosiglitazone or rosiglitazone and IGF1. RNA sequencing was used to determine the effect of wild-type IGF1 on gene expression in IHMGE spheroids. Figure 11A shows a flowchart of the study design. As shown in Figure 11B, treatment of IHMGE spheroids with wild-type IGF1 significantly upregulated four IGFBPs: IGFBP2, IGFBP5, IGFBP4, and IGFBP6. As shown in Figure 11B, among them, IGFBP2 was the most significantly upregulated IGFBP in response to wild-type IGF1 treatment. As shown in Figure 11C, treatment of IHMGE spheroids with wtIGF1 significantly upregulated genes involved in fatty acid transport, lipid synthesis, and meibomian gland formation. The gene set of interest was adopted from Butovich I., 163 Exp Eye Res. 2-16, 2017.

[0114] Example 5: In vivo effects of IGF-1 variants on mouse meibomian glands

[0115] material and method

[0116] Animal testing

[0117] Wild-type (WT) female C57BL / 6J mice (Jackson Labs, Strain #000664) were purchased at 2 months of age or 1–1.5 years of age. All animal studies were performed in accordance with the relevant protocols.

[0118] dosage

[0119] For the dosing test, mice were administered vehicle (endotoxin-free PBS, EMD Millipore, TMS-012-A) or 10 milligrams per kilogram (mg / kg) of IGF-1 LR3 (SEQ ID NO: 12) resuspended in vehicle. Animals were dosed using an insulin syringe (BD, 329424). Mice were intraperitoneally (IP) dosed with IGF-1 LR3 twice at 12-hour intervals, and sacrificed 24 or 48 hours after dosing as described below (results in Figures 14A and 14B). Mice were IP dosed daily with vehicle or 10 mg / kg of IGF-1 LR3 for 5 consecutive days, followed by 2 days of rest, for a total of 4 weeks, and then sacrificed as described below (results in Figures 15A, 15B, 16A, and 16B). Animals were given a single IP dose of 10 mg / kg IGF-1 LR3 or 10 μl eye drops of IGF-1 LR3 vehicle at a concentration of 5 mg / ml and then sacrificed as described below (results in Figure 17).

[0120] In Figure 18, animals were administered 10 μl of eye drops containing equimolar concentrations based on molecular weight of either 1 mg / ml wild-type IGF-1 (SEQ ID NO: 1) or 1.27 mg / ml IGF-1 LR3 (SEQ ID NO: 12), and then sacrificed as described above 0.5 or 2 hours after dosing. Eyelids were removed, frozen on dry ice, and then stored at -80°C until analysis by P-AKT ELISA.

[0121] In Figure 19, animals were administered 10 μl of eye drops containing equimolar concentrations based on molecular weight of either 1 mg / ml wild-type IGF-1 (SEQ ID NO: 1), 1.27 mg / ml IGF-1 LR3 (SEQ ID NO: 12), or 1 mg / mL rhIGF-1 Des1-3 (SEQ ID NO: 3), and then sacrificed as described above 2 hours after dosing. Eyelids were removed, frozen on dry ice, and then stored at -80°C until analysis by P-AKT ELISA.

[0122] In Figure 20, animals were administered 10 μl of eye drops containing equimolar concentrations based on molecular weight of either 1.27 mg / ml IGF-1 LR3 (SEQ ID NO: 12) or 1 mg / ml IGF-1 E3R (SEQ ID NO: 4), and then sacrificed as described above 1 hour after dosing. Eyelids were removed, frozen on dry ice, and then stored at -80°C until analysis by P-AKT ELISA.

[0123] In Figure 21, animals were administered 10 μl eye drops containing 0.3 mg / ml, 1 mg / ml, or 3 mg / ml IGF-1 LR3 (SEQ ID NO: 12) daily for two weeks and sacrificed 24 hours after the last dose.

[0124] In Figures 22A and 22B, animals received vehicle (PBS) or 1 mg / mL IGF-1 LR3 (SEQ ID NO: 12), 10 μL in each eye, daily in both eyes for 4 weeks.

[0125] In Figure 23, animals were administered 10 μl of eye drops containing equimolar concentrations based on molecular weight of either 1.27 mg / ml IGF-1 LR3 (SEQ ID NO: 12) or 1 mg / ml IGF-1 E3R (SEQ ID NO: 4) daily for two weeks and sacrificed 24 hours after the last administration.

[0126] Tissue collection, processing, and analysis

[0127] Mice were anesthetized with isoflurane and sacrificed by cervical dislocation, and the left and right upper eyelids were removed. The eyelids were then trimmed to a central 4 mm section of tissue, embedded in Tissue-Tek® OCT Compound (Sakura Finetek, Torrance, CA), and flash-frozen in liquid nitrogen. The tissue blocks were then sectioned at 8 μm thickness using a Leica CM 1850 cryostat (Leica, Wetzlar, Germany). The tissue sections were then stored in a deep freezer until processed for fluorescence microscopy.

[0128] To measure the basal proliferation rate, or the effect of IGF-1LR3 on cell proliferation (Figures 13A, 13B, 14A, and 14B), tissue sections were stained with Ki67, a cell proliferation marker. Rabbit anti-Ki67 (Abcam, catalog #ab15580) was reacted with tissue sections for 1 hour at 37°C in a humidified chamber. The sections were then washed with PBS (3 x 5 min), and secondary antibody (goat anti-rabbit AlexaFluor546, Invitrogen) was applied to the sections for 1 hour. The sections were then rinsed with PBS (3 x 5 min), counterstained with DAPI (1:5000), and then coverslipped. Fluorescence imaging was performed on a Leica DMI6000B fully automated inverted fluorescence microscope (Leica Microsystems Inc., Buffalo Grove, IL). Tile images across eyelid tissue sections and meibomian glands were collected using a low-light level camera (QIClick, QImaging, British Columbia, Canada) and a Leica 20x HC Plan Apo, 0.75 NA objective. Meta Imaging Series stitching was then performed. Quantification of Ki67 labeling was performed using QuPath, a bioimaging software analysis tool for high-throughput biomarker analysis. First, individual acini in each tissue section were annotated using the freeform region of interest tool, and Ki67-positive staining nuclei within each acinus were identified using the positive cell detection tool. The number of Ki67-positive cells and the perimeter of each acinus were then recorded, and the average number of labeled cells per 100 μm of acinar perimeter was calculated for each section. The average of three tissue sections was then determined, and the average for each mouse was calculated. Differences between treatment groups were then determined using Student's t test (Figures 13A and 13B) or two-way analysis of variance, Tukey's all pairwise multiple comparison procedure in both SigmaStat (Systat Software Inc., Point Richmond, CA) and GraphPad Prism (Insight Partners, New York, NY).

[0129] To assess changes in lipid synthesis (Figures 16A and 16B), cells were stained with HCS LipidTox (Invitrogen, Carlsbad, CA), a neutral lipid fluorescent probe. Cells were first fixed with 2% paraformaldehyde in PBS, then rinsed and incubated in LipidTox solution (1:100 dilution) at room temperature for 20 minutes, followed by DAPI staining. Fluorescent staining was then imaged using a Leica DMI6000B inverted microscope, and tiled images across the eyelid section were collected and stitched together using Meta Imaging software. To quantify newly synthesized lipid droplets near basal acinar cells, a threshold subroutine was used to segment the area of ​​lipid staining in the meibomian gland to identify pixels with an intensity greater than 50. Individual newly synthesized lipid droplets that showed high-intensity fluorescent staining were then identified using a counting subroutine set to extract threshold regions using the duplicate plane function and identify particles with diameters greater than 2 μm but less than 5 μm and a threshold intensity greater than 1000. Individual acini were then manually outlined using the freehand region tool, and the area of ​​the acini and the number of lipid droplets / acinus were recorded. The number of lipid droplets / acinus was then calculated based on the average acinar area of ​​4000 μm. 2 The mean number of lipid droplets in three individual sections was then calculated, followed by the mean number per mouse. Differences in the number of droplets / acini per treatment group were then determined using one-way analysis of variance, all pairwise multiple comparisons using Tukey's method in GraphPad Prism (Insight Partners, New York, NY).

[0130] To assess IGF1R1 activation by IGF-1 LR3 (Figure 17), phospho-AKT levels were measured using the phospho-Akt (S473) Pan Specific DuoSet IC ELISA (RnD #DYC887B-2). Eyelid tissues were flash-frozen on dry ice and stored at -80°C until further analysis. Samples were weighed and homogenized at 1 mL / 50 mg tissue in T-PER (Thermo Scientific, 78510) containing protease and phosphatase inhibitors (Thermo Scientific, A32959) using a NextAdvance Bullet Blender Gold (BB24AU) with Green, Navy, or Red Eppendorf Lysis Kits (NextAdvance). The homogenate was then used directly in the assay. ELISA was performed in 384-well plates following the manufacturer's recommendations with minor modifications. Briefly, wells of a 384-well plate were coated with 25 μl of 6 μg / ml phosphorylated Akt1 (S473) capture antibody (#841692) in PBS (25 μl per 384-well plate), sealed, and incubated overnight at room temperature. The wells were washed four times with 100 μl of PBS containing 0.05% Tween 20 and blocked with 50 μl of PBS containing 2% BSA for 1-2 hours at room temperature (RT). The wells were washed four times with 100 μl of PBS containing 0.05% Tween 20 and incubated with 25 μl of lysate or P-AKT standard for 2 hours at room temperature. Wells were washed four times with 100 μl of PBS containing 0.05% Tween 20 and then incubated for 1 hour at room temperature with 25 μl of phospho-Akt1 (S473) detection antibody (#843081) diluted at 100 nanograms per milliliter (ng / ml) in PBS containing 1% BSA. Wells were washed four times with 100 μl of PBS containing 0.05% Tween 20 and incubated for 20 minutes at room temperature with 25 μl of streptavidin-HRP A (RnD# 890803) diluted 1:200 in PBS containing 1% BSA.Wells were washed four times with 100 μl of PBS containing 0.05% Tween 20 and incubated with 25 μl of TMB substrate solution (Abcam TMB ELISA Substrate High Sensitivity (ab171523; lot GR3427893-1)) for 10–20 minutes at room temperature. 12.5 μl of stop solution (RnD# 895926 from Ancillary Kit 2) was added. Standard curves and OD450 data for each sample were collected using a Promega GloMAX Discover Microplate Reader. Sample data were interpolated from the standard curve, corrected for dilution factors, plotted, and pAKT levels were plotted and statistically compared using one-way ANOVA and Tukey's test for multiple comparisons in GraphPad Prism (Insight Partners, New York, NY).

[0131] Mouse eyelid meibography

[0132] Transillumination meibography (Figures 15A and 15B) was performed prior to the start of the study and immediately prior to sacrifice using previously published techniques. Briefly, mice were anesthetized using isoflurane and placed under a binocular dissecting microscope (Leica MZ16FA, Leica Microsystems, Heerbruck, Switzerland) equipped with a monochromatic camera (DFC340FX, Leica Microsystems, Heerbruck, Switzerland). A broadband halogen light source (OSL2 and OSL2B, Thorlabs, Newton, NJ) was used to transilluminate the eyelid for meibography. Light was delivered by a specialized optical fiber (BFL200HS02, Thorlabs, Newton, NJ) with an input connector consisting of a bundle of seven 200 μm-diameter fibers and an output connector consisting of a linear array of fibers adhesively attached to a diffuser and prism. All images were taken at 20x magnification. After sacrifice, ex vivo meibography was performed by removing the eyelids, placing them on an LED-backlit plate, and taking transillumination photographs using a dissecting microscope. To quantify meibomian gland area, individual eyelid images were analyzed using Meta Imaging Series software (Molecular Devices, Downingtown, PA). Specifically, the area of ​​each individual meibomian gland was manually outlined, and the area of ​​the target region was measured for each individual gland in each image. The mean gland area in each eyelid was then calculated, and differences between groups were statistically analyzed using a one-sided Mann-Whitney test using GraphPad Prism (Insight Partners, New York, NY).

[0133] Immunohistochemical staining (IHC)

[0134] Eyelids were dissected from wild-type (WT) female C57BL / 6J mice (Jackson Labs, Strain #000664). Tissues were fixed overnight in 4% paraformaldehyde and switched to 30% sucrose after 24 hours. Eyelids were then embedded in OCT medium, sectioned at 10 microns, and attached to glass slides. Tissues were then stained for Ki67 using 1 μg / mL rat anti-Ki67 (Invitrogen #14-5698-82) followed by detection with an anti-rat Alexafluor-conjugated secondary antibody. Tissues were also stained for IGFBP2 using 1 μg / mL rabbit anti-IGFBP2 (Abcam #ab188200) followed by detection with an anti-rabbit Alexafluor-conjugated secondary antibody. Between antibody incubations, slides were washed three times with PBS + 0.1% Tween-20. Slides were then cover-slipped and imaged.

[0135] In Figures 21 and 23, animals were sacrificed as described above, and eyelids were fixed in 4% paraformaldehyde overnight and switched to 30% sucrose after 24 hours. The eyelids were then embedded in OCT medium, sectioned at 10 microns, and attached to glass slides. The tissues were then stained for Ki67 using 1 μg / mL rat anti-Ki67 (Invitrogen #14-5698-82) and subsequently detected with an anti-rat Alexafluor-labeled secondary antibody. Between antibody incubations, the slides were washed three times with PBS + 0.1% Tween-20. The slides were then cover-slipped and imaged. Cell nuclei were then stained with Hoechst and covered with a cover-slip for imaging. Random regions of interest (ROIs) containing meibomian acini were identified. The perimeter of the acinus was outlined based on Hoechst staining in Image J, and the number of ki67+ cells along the perimeter was measured and expressed as the number of ki67+ cells per 100 m of acinar perimeter. Data were then quantified and analyzed in GraphPad Prism (Insight Partners, New York, NY).

[0136] result

[0137] As shown in Figures 13A and 13B, young mice have increased proliferation in meibomian acini compared with old mice. Systemic IGF-1 LR3 treatment increased proliferation in meibomian acini of old mice (Figures 14A and 14B), reversed atrophy and increased meibomian gland area in old mice (Figures 15A and 15B), increased lipid synthesis in meibomian glands regardless of age (Figures 16A and 16B), and was able to activate IGF1R in the eyelids when delivered systemically or via eye drops (Figure 17).

[0138] Next, we investigated the duration of the effects of IGF-1 mutations. As shown in Figure 18, IGF-1 LR3 (SEQ ID NO: 12) maintained significantly elevated pAKT (IGF1R activation) 2 hours after administration, while wild-type IGF-1 no longer showed significant pAKT 2 hours after administration. As shown in Figure 19, IGF-1 LR3 (SEQ ID NO: 12) and another IGFBP-binding-deficient IGF-1 variant, IGF-1 Des1-3 (SEQ ID NO: 3), significantly elevated pAKT (IGF1R activation) 2 hours after administration compared with wild-type IGF-1. This suggests that some IGF-1 variants with reduced affinity for IGFBPs prolong the duration of IGF1R activation compared with wild-type IGF-1. Furthermore, IGF-1 LR3 (SEQ ID NO: 12) and IGF-1 E3R (SEQ ID NO: 4) have similar potencies in vivo. As shown in Figure 20, IGF-1 LR3 (SEQ ID NO: 12) or the IGFBP-binding-deficient IGF-1 variant IGF-1 E3R (SEQ ID NO: 4) elevated pAKT (IGF1R activation) to levels comparable to IGF-1 at 1 hour post-dosing.

[0139] Next, we tested the effects of IGF-1 LR3 and IGF-1 ER3 on the meibomian glands of aged mice. As shown in Figure 21, IGF-1 LR3 (SEQ ID NO: 12) induced dose-responsive proliferation in basal cells of the meibomian glands. As shown in Figure 21, daily eye drop administration of IGF-1 LR3 (SEQ ID NO: 12) at set concentrations of 0.3, 1, or 3 mg / ml for two weeks induced basal cell proliferation as determined by IHC. IGF-1 LR3 (SEQ ID NO: 12) regenerated meibomian gland atrophy in aged mice. As shown in Figures 22A and 22B, daily treatment with IGF-1 LR3 (SEQ ID NO: 12) for one month increased the area of ​​the meibomian glands, comparing pre- and post-treatment. IGF-1 LR3 (SEQ ID NO: 12) and IGF-1 E3R (SEQ ID NO: 4) induced proliferation of basal cells in the meibomian glands. As shown in Figure 23, daily eye drop administration of IGF-1 LR3 (SEQ ID NO: 12) or IGF-1 E3R (SEQ ID NO: 4) for two weeks induced basal cell proliferation in aged mice as determined by IHC.

[0140] While preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous modifications, changes, and substitutions will occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. The following claims define the scope of the invention, and it is intended that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

1. A pharmaceutical composition comprising a therapeutically effective amount of an IGF-1 variant that has reduced affinity for at least one IGF binding protein (IGFBP) compared to the affinity for the interaction between wild-type IGF-1 (SEQ ID NO: 1) and the IGFBP, wherein the pharmaceutical composition is formulated for topical administration.

2. 10. The pharmaceutical composition of claim 1, formulated for topical administration to the eye or eyelid.

3. 1. A pharmaceutical composition comprising a therapeutically effective amount of an IGF-1 variant, wherein topical administration of said pharmaceutical composition to the eye or eyelid comprises: (a) an increase in the size of the meibomian glands; (b) reduced meibomian gland atrophy; (c) Reversal of age-related meibomian gland atrophy, (d) increasing the function of one or more meibocytes; (e) increased corneal epithelial cell proliferation; (f) increased corneal healing rate; (g) increasing IGF1 receptor (IGF1R) activation in the meibomian glands; (h) increasing the duration of IGF1R activation in the meibomian glands; and (i) increasing the lipid content of the meibomian glands; A pharmaceutical composition providing one or more of:

4. 4. The pharmaceutical composition of claim 3, wherein the IGF-1 variant has reduced affinity for at least one IGF binding protein compared to the affinity of wild-type IGF-1 for the IGFBP.

5. 5. The pharmaceutical composition of any one of claims 1 to 4, wherein the IGF-1 variant has at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to SEQ ID NO:

1.

6. The pharmaceutical composition of any one of claims 1 to 5, formulated for delivery by eye drops.

7. 6. The pharmaceutical composition of any one of claims 1 to 5, comprising a cream for administration to one or both eyelids of a subject.

8. 8. The pharmaceutical composition of any one of claims 1 to 7, which, when administered to a subject suffering from meibomian gland dysfunction, results in an increase in the median surface area or volume of the meibomian glands within the inner eyelid surface of the subject compared to a subject not administered the pharmaceutical composition.

9. 9. The pharmaceutical composition of any one of claims 1 to 8, which, when administered to a subject suffering from meibomian gland dysfunction, results in an increase in the median lipid content in the meibomian glands of the subject compared to a subject not administered the pharmaceutical composition.

10. 10. The pharmaceutical composition of any one of claims 1 to 9, which, when administered to a subject suffering from meibomian gland dysfunction, results in an increase in median lipid quality in the meibomian glands of the subject compared to a subject not administered the pharmaceutical composition.

11. 11. The pharmaceutical composition of any one of claims 1 to 10, which, when administered to a subject suffering from meibomian gland dysfunction, results in an increase in median lipid release from acini in the meibomian glands of the subject compared to a subject not administered the pharmaceutical composition.

12. 12. The pharmaceutical composition of any one of claims 1 to 11, which, when administered to a subject suffering from meibomian gland dysfunction, results in an increase in the median duration of Akt phosphorylation in meibocytes compared to a subject not administered the pharmaceutical composition.

13. The pharmaceutical composition according to any one of claims 1 to 12, wherein the IGF-1 variant is a truncation.

14. The pharmaceutical composition according to any one of claims 1 to 12, wherein said IGF-1 variant comprises or consists of the amino acid sequence of SEQ ID NO:

3.

15. The pharmaceutical composition of any one of claims 1 to 13, wherein said IGF-1 variant comprises one or more amino acid substitutions relative to wild-type IGF-1 (SEQ ID NO: 1).

16. 16. The pharmaceutical composition of any one of claims 1 to 13 and 15, wherein the IGF-1 variant comprises an amino acid deletion at position 37 relative to wild-type IGF-1 (SEQ ID NO: 1), wherein the numbering of positions is based on an alignment of the IGF-1 variant with SEQ ID NO: 1, starting from position 1 at the N-terminus, and numbering positions from the N-terminus of SEQ ID NO: 1 to the C-terminus of SEQ ID NO:

1.

17. The pharmaceutical composition of any one of claims 1 to 13, 15 and 16, wherein the IGF-1 variant comprises the amino sequence of SEQ ID NO:

6.

18. 15. The pharmaceutical composition of any one of claims 1 to 12 and 14, wherein the IGF-1 variant comprises an amino acid substitution at position 60 relative to wild-type IGF-1 (SEQ ID NO: 1), wherein the numbering of positions is based on an alignment of the IGF-1 variant with SEQ ID NO: 1, starting from position 1 at the N-terminus, and numbering positions from the N-terminus of SEQ ID NO: 1 to the C-terminus of SEQ ID NO:

1.

19. 19. The pharmaceutical composition of any one of claims 1 to 12, 15, or 18, wherein the IGF-1 variant comprises the amino sequence of SEQ ID NO:

2.

20. 15. The pharmaceutical composition of any one of claims 1 to 12 and 14, wherein the IGF-1 variant comprises an amino acid substitution at position 3 relative to wild-type IGF-1 (SEQ ID NO: 1), wherein the numbering of positions is based on an alignment of the IGF-1 variant with SEQ ID NO: 1, starting from position 1 at the N-terminus and numbering from the N-terminus of SEQ ID NO: 1 to the C-terminus of SEQ ID NO:

1.

21. 21. The pharmaceutical composition of any one of claims 1 to 12, 15, or 20, wherein the IGF-1 variant comprises the amino sequence of SEQ ID NO:

4.

22. 16. The pharmaceutical composition of any one of claims 1 to 12 or 15, wherein said IGF-1 variant comprises the amino sequence of SEQ ID NO:

5.

23. The pharmaceutical composition of any one of claims 1 to 12, wherein said IGF-1 variant comprises the amino acid sequence of SEQ ID NO:

7.

24. The pharmaceutical composition of any one of claims 1 to 12, wherein said IGF-1 variant comprises the amino acid sequence of SEQ ID NO:

12.

25. The pharmaceutical composition of any one of claims 1 to 24, wherein the IGF-1 variant is conjugated to a cell-penetrating peptide (CPP) or a skin-penetrating peptide (SPP).

26. 26. The pharmaceutical composition of claim 25, wherein the IGF-I variant is linked to a cell-penetrating peptide selected from the group consisting of SEQ ID NO: 10 and SEQ ID NO:

11.

27. 26. The pharmaceutical composition of claim 25, wherein the IGF-1 variant is linked to a skin-penetrating peptide of SEQ ID NO:

9.

28. 28. The pharmaceutical composition of claim 27, wherein the IGF-1 variant comprises the amino acid sequence of SEQ ID NO:

8.

29. 29. The pharmaceutical composition of any one of claims 1 to 28, further comprising one or more pharmaceutically acceptable excipients.

30. 30. The pharmaceutical composition of claim 29, wherein the one or more pharmaceutically acceptable excipients comprise one or more of water, saline, sucrose, lactose, malic acid, cellulose sugars, mannitol, maltitol, dextran, sorbitol, starch, agar, alginate, chitin, chitosan, pectin, gum tragacanth, gum arabic, gelatin, collagen, casein, albumin, synthetic or semi-synthetic polymers or glycerides, methylcellulose, hydroxypropylmethylcellulose, and polyvinylpyrrolidone.

31. 31. The pharmaceutical composition of any one of claims 1-2 and 4-30, wherein the at least one IGFBP comprises IGFBP2.

32. 33. The pharmaceutical composition of any one of claims 1-2 and 4-32, wherein the at least one IGFBP comprises IGFBP3.

33. 32. The pharmaceutical composition of any one of claims 1-2 and 4-31, wherein the at least one IGFBP comprises IGFBP1.

34. 33. The pharmaceutical composition of any one of claims 1-2 and 4-32, wherein the at least one IGFBP comprises IGFBP4.

35. 33. The pharmaceutical composition of any one of claims 1-2 and 4-32, wherein the at least one IGFBP comprises IGFBP5.

36. 33. The pharmaceutical composition of any one of claims 1-2 and 4-32, wherein the at least one IGFBP comprises IGFBP6.

37. 37. The pharmaceutical composition of any one of claims 1 to 36, wherein administration of the pharmaceutical composition to the eye or eyelid of the subject results in an increase in the size of the meibomian gland.

38. 38. The pharmaceutical composition of any one of claims 1 to 37, wherein administration of the pharmaceutical composition to the eye or eyelid of the subject results in a reduction in meibomian gland atrophy.

39. 39. The pharmaceutical composition of any one of claims 1 to 38, wherein administration of the pharmaceutical composition to the eye or eyelid of the subject results in the reversal of age-related meibomian gland atrophy.

40. 40. The pharmaceutical composition of any one of claims 1 to 39, wherein administration of the pharmaceutical composition to the eye or eyelid of the subject results in an increase in the function of one or more meibocytes.

41. 41. The pharmaceutical composition of any one of claims 1 to 40, wherein administration of the pharmaceutical composition to the eye or eyelid of the subject results in increased proliferation of corneal epithelial cells.

42. 42. The pharmaceutical composition of any one of claims 1 to 41, wherein administration of the pharmaceutical composition to the eye or eyelid of the subject results in an increased rate of corneal healing.

43. 43. The pharmaceutical composition of any one of claims 1 to 42, wherein administration of the pharmaceutical composition to the eye or eyelid of the subject results in increased IGF1 receptor (IGF1R) activation in the meibomian gland.

44. 44. The pharmaceutical composition of any one of claims 1 to 43, wherein administration of the pharmaceutical composition to the eye or eyelid of the subject results in an increased duration of IGF1R activation in the meibomian gland.

45. 45. The pharmaceutical composition of any one of claims 1 to 44, wherein administration of the pharmaceutical composition to the eye or eyelid of the subject results in an increase in the lipid content of the meibomian gland.

46. 46. ​​The pharmaceutical composition of any one of claims 1 to 45, which, when administered to spheroids of IHGMGE cells, results in an increase in the lipid content in said spheroids on average.

47. (a) a pharmaceutical composition according to any one of claims 1 to 6 and 1 to 46; (b) an eye dropper for delivering the pharmaceutical composition as an eye drop; Kit including:

48. 1. A method for treating an ocular disorder in a subject in need thereof, comprising administering to a subject having the ocular disorder a pharmaceutical composition, wherein the pharmaceutical composition comprises a therapeutically effective amount of an IGF-1 variant that has reduced affinity for an IGF-binding protein (IGFBP) compared to the affinity for the interaction between wild-type IGF-1 (SEQ ID NO: 1) and the IGFBP.

49. 1. A method for locally administering an IGF-1 variant to a subject, the method comprising the step of administering an IGF-1 variant to a subject, wherein the IGF-1 variant has reduced affinity for an IGF binding protein (IGFBP) compared to the affinity of the interaction between wild-type IGF-1 (SEQ ID NO: 1) and the IGFBP.

50. The method according to claim 48 or 49, wherein the pharmaceutical composition is a pharmaceutical composition according to any one of claims 1 to 46.

51. 51. The method of any one of claims 48 to 50, wherein the pharmaceutical composition is administered to the eye or eyelid of the subject.

52. 52. The method of claim 51, wherein the pharmaceutical composition is administered to the eye of the subject by eyedropper.

53. 52. The method of any one of claims 48 to 51, wherein the pharmaceutical composition is administered to the outer eyelid of the subject.

54. 54. The method of claim 53, wherein the pharmaceutical composition is a cream.

55. 55. The method of any one of claims 48 to 54, wherein the pharmaceutical composition is administered to a subject suffering from meibomian gland dysfunction.

56. 56. The method of any one of claims 48 to 55, wherein administering the pharmaceutical composition to the subject results in an increase in the surface area or volume of the meibomian glands within the inner eyelid surface of the subject.

57. 57. The method of any one of claims 48 to 56, wherein administering the pharmaceutical composition to the subject results in an increase in lipid content in the meibomian glands of the subject.

58. 58. The method of any one of claims 48 to 57, wherein administering the pharmaceutical composition to the subject results in increased release of lipids from the acini of the meibomian glands of the subject.

59. 59. The method of any one of claims 48 to 58, wherein administering the pharmaceutical composition to the subject results in an increase in the duration of Akt phosphorylation in meibocytes.

60. 60. The method of any one of claims 48 to 59, which does not include the administration of any additional phospholipidosis-inducing agent.

61. 61. The method of any one of claims 48 to 60, which does not include administration of one or both of azithromycin and doxycycline.

62. 62. The method of any one of claims 48 or 50-61, wherein the eye disorder comprises dry eye disease.

63. 63. The method of any one of claims 48 or 50-62, wherein the eye disorder comprises meibomian gland dysfunction.

64. 64. The method of any one of claims 48 or 50-63, wherein the eye disorder comprises Sjogren's syndrome.

65. (a) a therapeutically effective amount of a polypeptide comprising the amino acid sequence of any one of SEQ ID NOs: 1-8 or 12; (b) one or more pharmaceutically acceptable excipients; and A pharmaceutical composition comprising:

66. 66. The pharmaceutical composition of claim 65, which is a solution for delivery as eye drops.

67. 66. The pharmaceutical composition of claim 65, comprising a cream for administration to one or both eyelids.

68. 66. The pharmaceutical composition of claim 65, formulated for systemic delivery.

69. 69. The pharmaceutical composition of any one of claims 65 to 68, wherein the polypeptide is a human IGF-1R agonist.

70. 70. The pharmaceutical composition of any one of claims 65 to 69, wherein administration of the pharmaceutical composition to a subject suffering from meibomian gland dysfunction results in an increase in the median surface area or volume of the meibomian glands within the inner eyelid surface.

71. 71. The pharmaceutical composition of any one of claims 65 to 70, wherein administration of the pharmaceutical composition to a subject suffering from meibomian gland dysfunction results in an increase in the median lipid content in the meibomian glands.

72. 72. The pharmaceutical composition of any one of claims 65 to 71, wherein administration of the pharmaceutical composition to a subject suffering from meibomian gland dysfunction results in an increase in median lipid release from the acini of the meibomian glands.

73. 73. The pharmaceutical composition of any one of claims 65 to 72, wherein administration of the pharmaceutical composition to a subject suffering from meibomian gland dysfunction results in an increase in median release of lipids from the acini of the meibomian glands.

74. 74. The pharmaceutical composition of any one of claims 65 to 73, wherein administration of the pharmaceutical composition to a subject suffering from meibomian gland dysfunction results in an increase in median phosphorylation of Akt in meibocytes.

75. 75. The pharmaceutical composition according to any one of claims 65 to 74, which does not contain any additional phospholipidosis-inducing agent.

76. 76. The pharmaceutical composition of any one of claims 65 to 75, which does not contain either azithromycin or doxycycline.

77. 77. The pharmaceutical composition of any one of claims 65-76, wherein the one or more pharmaceutically acceptable excipients comprise one or more of water, saline, sucrose, lactose, malic acid, cellulose sugars, mannitol, maltitol, dextran, sorbitol, starch, agar, alginate, chitin, chitosan, pectin, gum tragacanth, gum arabic, gelatin, collagen, casein, albumin, synthetic or semi-synthetic polymers or glycerides, methylcellulose, hydroxypropylmethylcellulose, and polyvinylpyrrolidone.

78. 78. The pharmaceutical composition of any one of claims 65 to 77, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO:

1.

79. 78. The pharmaceutical composition of any one of claims 65 to 77, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO:

2.

80. 78. The pharmaceutical composition of any one of claims 65 to 77, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO:

3.

81. 78. The pharmaceutical composition of any one of claims 65 to 77, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO:

4.

82. 78. The pharmaceutical composition of any one of claims 65 to 77, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO:

5.

83. 78. The pharmaceutical composition of any one of claims 65 to 77, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO:

6.

84. 78. The pharmaceutical composition of any one of claims 65 to 77, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO:

7.

85. 78. The pharmaceutical composition of any one of claims 65 to 77, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO:

8.

86. 78. The pharmaceutical composition of any one of claims 65 to 77, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO:

12.

87. 87. The pharmaceutical composition of any one of claims 1 to 86, wherein the polypeptide further comprises a cell-penetrating peptide (CPP) or a skin-penetrating peptide (SPP).

88. 88. The pharmaceutical composition of claim 87, wherein the polypeptide comprises a cell-penetrating peptide selected from the group consisting of SEQ ID NO: 10 and SEQ ID NO:

11.

89. 89. The pharmaceutical composition of any one of claims 1 to 88, wherein the polypeptide comprises a skin-penetrating peptide of SEQ ID NO:

9.

90. (a) a pharmaceutical composition according to any one of claims 65-66 and 68-89; (b) an eye dropper for delivering the pharmaceutical composition as an eye drop; Kit including:

91. 1. A method of treating an ocular disorder in a subject in need thereof, comprising: A method comprising the step of administering to said subject a pharmaceutical composition comprising any one of SEQ ID NOs: 1-8 or 12.

92. The method of claim 91, wherein the pharmaceutical composition is a pharmaceutical composition according to any one of claims 65 to 91.

93. 93. The method of claim 91 or 92, wherein the pharmaceutical composition is administered to the eye of the subject.

94. 94. The method of claim 93, wherein the pharmaceutical composition is administered to the eye of the subject by eyedropper.

95. 93. The method of claim 91 or 92, wherein the pharmaceutical composition is administered to the outer eyelid of the subject.

96. 96. The method of claim 95, wherein the pharmaceutical composition is a cream.

97. 97. The method of any one of claims 91 to 96, wherein administering the pharmaceutical composition to the subject increases the median surface area or volume of the meibomian glands on the inner eyelid surface.

98. 98. The method of any one of claims 91 to 97, wherein administering the pharmaceutical composition to the subject results in an increase in lipid content in the meibomian glands.

99. 99. The method of any one of claims 91 to 98, wherein administration of the pharmaceutical composition to the subject suffering from meibomian gland dysfunction results in increased release of lipids from the acini of the meibomian glands.

100. 100. The method of any one of claims 91 to 99, wherein administering the pharmaceutical composition to the subject results in increased release of lipids from the acini of the meibomian glands.

101. The method of any one of claims 91 to 100, wherein administering the pharmaceutical composition to the subject results in increased phosphorylation of Akt in meibocytes.

102. 102. The method of any one of claims 91 to 101, which does not comprise the administration of any additional phospholipidosis-inducing agent.

103. 103. The method of any one of claims 91 to 102, which does not include administration of either azithromycin or doxycycline.

104. 104. The method of any one of claims 91 to 103, wherein the eye disorder comprises dry eye disease.

105. 104. The method of any one of claims 91 to 103, wherein the eye disorder comprises meibomian gland dysfunction.

106. 104. The method of any one of claims 91 to 103, wherein the eye disorder comprises Sjogren's syndrome.

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