Compositions and methods for treating meibomian gland dysfunction

Abstract

The present disclosure provides compositions for use in the treatment of eye-related conditions (e.g., conditions that affect the meibomian gland). Compositions for use in such conditions can act on the IGF-1 pathway, such as by binding (e.g., agonizing) IGF1R. Other compositions may act on the IGF-1 pathway by other mechanisms. The present disclosure also discloses related methods, kits, and pharmaceutical compositions for treating eye-related conditions.

Description

[0001] Cross-references

[0002] 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.

[0003] Sequence Listing

[0004] This application contains a sequence listing submitted electronically in XML file format and is incorporated herein by reference in its entirety. The XML copy was created on December 21, 2023, is named 61766-715_601_SL.xml, and is 12,698 bytes in size. Background Art

[0005] Meibomian glands are oil glands located primarily at the margins of the upper and lower eyelids. These glands play an important role in preventing dry eyes: they secrete meibum, a mixture of lipids and proteins that coats the surface of the eye, and they prevent the evaporation of the aqueous component of tears. Meibomian gland dysfunction and morphological changes can lead to dry eye disease (DED). There are no approved medications for the treatment of meibomian gland dysfunction (MGD). While some medications provide some symptom relief, there are currently no disease-modifying therapies. Approved medications target inflammation or promote the production of low-quality aqueous tears, but do not provide any long-term improvement for patients. Furthermore, these therapies require frequent dosing and high patient compliance to be effective. Summary of the Invention

[0006] In one aspect, the present disclosure provides a pharmaceutical composition comprising a therapeutically effective amount of an IGF-1 variant having reduced affinity for at least one IGF binding protein (IGFBP) compared to the affinity of the interaction between wild-type IGF-1 (SEQ ID NO: 1) and the IGFBP, wherein the pharmaceutical composition is formulated for topical administration. In some embodiments, the pharmaceutical composition is formulated for topical administration to the eye or eyelid.

[0007] In another aspect, the present disclosure provides a pharmaceutical composition comprising a therapeutically effective amount of an IGF-1 variant, wherein topical application of the pharmaceutical composition to the eye or eyelid results in one or more of: an increase in the size of the meibomian glands; a decrease in meibomian gland atrophy; a reversal of age-related meibomian gland atrophy; an increase in the function of one or more meibomian gland cells; an increase in corneal epithelial cell proliferation; an increase in the rate of corneal healing; an increase in IGF1 receptor (IGF1R) activation in the meibomian glands; an increase in the duration of IGF1R activation in the meibomian glands; and an increase in the lipid content of the meibomian glands.

[0008] In some embodiments, the IGF-1 variant has reduced affinity for at least one IGF binding protein relative to the affinity of wild-type IGF-1 for the 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 via eye drops. In some embodiments, the pharmaceutical composition comprises a cream for application to one or both eyelids of a subject. In some embodiments, when the pharmaceutical composition is administered to a subject with meibomian gland dysfunction, the pharmaceutical composition 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 who did not receive the pharmaceutical composition. In some embodiments, when the pharmaceutical composition is administered to a subject with meibomian gland dysfunction, the pharmaceutical composition results in an increase in the median lipid content within the meibomian glands of the subject compared to a subject who did not receive the pharmaceutical composition. In some embodiments, when the pharmaceutical composition is administered to a subject with meibomian gland dysfunction, the pharmaceutical composition results in an increase in the median lipid mass within the subject's meibomian glands compared to a subject that did not receive the pharmaceutical composition. In some embodiments, when the pharmaceutical composition is administered to a subject with meibomian gland dysfunction, the pharmaceutical composition results in an increase in the median lipid release from the acini of the subject's meibomian glands compared to a subject that did not receive the pharmaceutical composition. In some embodiments, when the pharmaceutical composition is administered to a subject with meibomian gland dysfunction, the pharmaceutical composition results in an increase in the median duration of Akt phosphorylation in meibomian gland cells relative to a subject that did not receive the pharmaceutical composition. In some embodiments, the IGF-1 variant is a truncated form. 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), wherein position numbering is based on alignment of the IGF-1 variant with SEQ ID NO: 1, wherein positions are numbered from the N-terminus of SEQ ID NO: 1 to the C-terminus of SEQ ID NO: 1, starting at position 1 of the N-terminus. 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), wherein position numbering is based on an alignment of the IGF-1 variant with SEQ ID NO: 1, wherein positions are numbered from the N-terminus of SEQ ID NO: 1 to the C-terminus of SEQ ID NO: 1, starting at position 1 of the N-terminus. 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), wherein position numbering is based on an alignment of the IGF-1 variant with SEQ ID NO: 1, wherein positions are numbered from the N-terminus of SEQ ID NO: 1 to the C-terminus of SEQ ID NO: 1, starting at position 1 of the N-terminus. In some embodiments, the IGF-1 variant comprises the amino acid sequence of SEQ ID NO: 4. In some embodiments, the IGF-1 variant comprises the amino acid sequence of SEQ ID NO: 5. In some embodiments, the IGF-1 variant comprises the amino acid sequence of SEQ ID NO: 7. In some embodiments, the IGF-1 variant comprises the amino acid sequence of SEQ ID NO: 12. In some embodiments, the IGF-1 variant is conjugated to a cell penetrating peptide (CPP) or a skin penetrating peptide (SPP). In some embodiments, the IGF-1 variant is conjugated to a cell penetrating peptide selected from SEQ ID NO: 10 and SEQ ID NO: 11. In some embodiments, the IGF-1 variant is conjugated to a skin penetrating peptide of SEQ ID NO: 9. In some embodiments, the IGF-1 variant comprises the amino acid 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, cellulosic sugars, mannitol, maltitol, dextran, sorbitol, starch, agar, alginate, chitin, chitosan, pectin, tragacanth, gum arabic, gelatin, collagen, casein, albumin, synthetic or semisynthetic polymers or glycerides, methylcellulose, hydroxypropyl methylcellulose, 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, administering the pharmaceutical composition to the eye or eyelid of the subject results in an increase in the size of the meibomian glands. In some embodiments, administering the pharmaceutical composition to the eye or eyelid of the subject results in a reduction in meibomian gland atrophy. In some embodiments, administering the pharmaceutical composition to the eye or eyelid of the subject results in a reversal of age-related meibomian gland atrophy. In some embodiments, administering the pharmaceutical composition to the eye or eyelid of the subject results in an increase in the function of one or more meibomian gland cells. In some embodiments, administering the pharmaceutical composition to the eye or eyelid of the subject results in an increase in corneal epithelial cell proliferation. In some embodiments, administering the pharmaceutical composition to the eye or eyelid of the subject results in an increase in corneal healing. In some embodiments, administering the pharmaceutical composition to the eye or eyelid of the subject results in an increase in IGF1 receptor (IGF1R) activation in the meibomian glands. In some embodiments, administering the pharmaceutical composition to the eye or eyelid of the subject results in an increase in the duration of IGF1R activation in the meibomian glands. In some embodiments, administering the pharmaceutical composition to the eye or eyelid of the subject results in an increase in lipid content of the meibomian glands. In some embodiments, when the pharmaceutical composition is administered to spheroids of IHGMGE cells, the pharmaceutical composition results in an increase in lipid content in the spheroids on average.

[0009] In another aspect, the present disclosure provides a kit comprising the pharmaceutical composition provided herein; and an eye dropper for delivering the pharmaceutical composition as an eye drop solution.

[0010] In another aspect, the present disclosure provides a method for treating an eye condition in a subject in need thereof, the method comprising administering a pharmaceutical composition to a subject suffering from an eye condition, wherein the pharmaceutical composition comprises a therapeutically effective amount of an IGF-1 variant having reduced affinity for an IGF binding protein (IGFBP) relative to the affinity of the interaction between wild-type IGF-1 (SEQ ID NO: 1) and the 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 eye dropper. 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 on the inner eyelid surface of the subject. In some embodiments, administering the pharmaceutical composition to the subject results in an increase in the lipid content within the subject's meibomian glands. In some embodiments, administering the pharmaceutical composition to the subject results in increased lipid release from the alveoli of the subject's meibomian glands. In some embodiments, administering the pharmaceutical composition to the subject results in increased duration of Akt phosphorylation in meibomian gland cells. In some embodiments, the method does not include administering any other phospholipid deposition inducer. In some embodiments, the method does not include administering one or both of azithromycin and doxycycline. In some embodiments, the eye condition comprises dry eye. In some embodiments, the eye condition comprises meibomian gland dysfunction. The eye condition comprises Sjögren's syndrome.

[0011] In another aspect, the present disclosure provides a pharmaceutical composition comprising: a therapeutically effective amount of a polypeptide comprising any one of the amino acid sequences 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 application to one or both eyelids. In some embodiments, the pharmaceutical composition is formulated for systemic delivery. In some embodiments, the polypeptide is a human IGF1R agonist. In some embodiments, administration of the pharmaceutical composition to a subject with meibomian gland dysfunction results in a median increase in the surface area or volume of the meibomian glands within the inner eyelid surface. In some embodiments, administration of the pharmaceutical composition to a subject with meibomian gland dysfunction results in an increase in the median lipid content within the meibomian glands. In some embodiments, administration of the pharmaceutical composition to a subject with meibomian gland dysfunction results in an increase in the median lipid release from the alveoli of the meibomian glands. In some embodiments, administration of the pharmaceutical composition to a subject with meibomian gland dysfunction results in an increase in the median lipid release from the alveoli of the meibomian glands. In some embodiments, administration of the pharmaceutical composition to subjects with meibomian gland dysfunction results in an increase in the median phosphorylation of Akt in meibomian gland cells. In some embodiments, the pharmaceutical composition does not contain any other phospholipid deposition inducer. In some embodiments, the pharmaceutical composition does not contain either azithromycin or doxycycline. In some embodiments, the one or more pharmaceutically acceptable excipients comprise one or more of water, saline, sucrose, lactose, malic acid, cellulosic sugars, mannitol, maltitol, dextran, sorbitol, starch, agar, alginate, chitin, chitosan, pectin, tragacanth, gum arabic, gelatin, collagen, casein, albumin, synthetic or semi-synthetic polymers or glycerides, methylcellulose, hydroxypropyl methylcellulose, and polyvinylpyrrolidone. In some embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NO: 2. In some embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NO: 3. In some embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NO: 4. In some embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NO: 5. In some embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NO: 6. In some embodiments, the polypeptide comprises the amino acid 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 acid 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 SEQ ID NO: 10 and SEQ ID NO: 11. In some embodiments, the polypeptide comprises a skin penetrating peptide of SEQ ID NO: 9.

[0012] In another aspect, the present disclosure provides a method for treating an eye condition 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 subject's eye. In some embodiments, the pharmaceutical composition is administered to the subject's eye via an eye dropper. 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, administering the pharmaceutical composition to the subject results in an increase in the median surface area or volume of the meibomian glands on the inner eyelid surface. In some embodiments, administering the pharmaceutical composition to the subject results in an increase in lipid content within the meibomian glands. In some embodiments, administering the pharmaceutical composition to the subject having meibomian gland dysfunction results in an increase in lipid release from the alveoli of the meibomian glands. In some embodiments, administering the pharmaceutical composition to the subject results in an increase in lipid release from the alveoli of the meibomian glands. In some embodiments, administering the pharmaceutical composition to the subject results in an increase in Akt phosphorylation in meibomian gland cells. In some embodiments, the method does not include the administration of any other phospholipid deposition inducing agent. In some embodiments, the method does not include the administration of either azithromycin or doxycycline. In some embodiments, the eye condition comprises dry eye. In some embodiments, the eye condition comprises meibomian gland dysfunction. In some embodiments, the eye condition comprises Sjögren's syndrome.

[0013] Other aspects and advantages of the present disclosure will become readily apparent to those skilled in the art from the following detailed description, wherein only exemplary embodiments of the present disclosure are shown and described. It should be understood that the present disclosure is susceptible of other and different embodiments, and that its several details may be modified 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 restrictive.

[0014] Incorporation by reference

[0015] 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 an incorporated-by-reference publication, patent, or patent application conflicts with the disclosure contained in the specification, the specification is intended to supersede and / or take precedence over any such conflicting material. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The novel features of the present invention are particularly set forth in the appended claims. The features and advantages of the present invention will be better understood by reference to the following detailed description of exemplary embodiments utilizing the principles of the present invention and the accompanying drawings, in which:

[0017] Figure 1 Depicted are the dose-dependent effects of IGF-1 (SEQ ID NO: 1), IGF-1Ea (SEQ ID NO: 7), IGF-1Des1-3 R37X (SEQ ID NO: 6), IGF-1Des1-3 (SEQ ID NO: 8), and IGF-1E3R (SEQ ID NO: 4) on AKT S473 phosphorylation in DU145 cells. Wild-type IGF-1 and IGF-1 variants have similar EC values ​​in DU145 cells. 50 value.

[0018] Figure 2 The affinity of IGFBPs for various IGF-1 mutants has been shown to be reduced. In L6 rat myoblast conditioned medium containing various IGF-binding proteins, competition assays reported that the affinity of BPs for IGF-1LR3 (SEQ ID NO: 12), LG3 (long IGF-1E3G), long IGF-1, and IGF-1Des1-3 (SEQ ID NO: 3) was reduced by a factor of 690, a factor of 112, a factor of 5.5, and a factor of 38, respectively (see Francis, GL et al., 8(3) J. Mol. Endocrinol. 213-223, 1992). In another study using bovine IGFBP2, the binding affinity of IGF-1E3R (SEQ ID NO: 4) and IGF E3G for IGFBPs was reported to be reduced by a factor of 230 and a factor of 59, respectively (see King, R. et al., 8 J. Mol. Endocrinol. 29-41, 1992).

[0019] Figure 3A and Figure 3BProvided are that IGF-1LR3 (SEQ ID NO: 12) and IGF-1E3R (SEQ ID NO: 4) are less inhibited by IGFBP2 and IGFBP3 than by wild-type IGF-1 (SEQ ID NO: 1). Figure 3A Depicted are the effects of wild-type IGF-1, IGF-1LR3, and IGF-1 E3R on AKT S473 phosphorylation in DU145 cells in the presence of IGFBP2 at an IGF-1 to IGFBP2 ratio of 1:1, 1:2, or 1:4, respectively. Figure 3B Depicted are the effects of wild-type IGF-1, IGF-1LR3, and IGF-1 E3R on AKT S473 phosphorylation in DU145 cells in the presence of IGFBP3 at an IGF-1 to IGFBP3 ratio of 1:1, 1:2, or 1:4, respectively.

[0020] Figure 4 The dose-dependent effect of IGF-1 on AKT S473 phosphorylation in immortalized human meibomian gland epithelial cells (IHMGEC) is depicted. 50 The value is approximately 0.07 nanomolar (nM).

[0021] Figure 5 Depicted are the results of a live cell imaging assay: upon IGF-1 stimulation, IHMGECs spread and proliferate at an increasing rate in a dose-dependent manner.

[0022] Figure 6 Depicted are the results of human IGF-1 stimulation in IGHMECs following a cell viability assay: cells proliferated in a dose-dependent manner in response to IGF-1.

[0023] Figure 7A and Figure 7B Depicts immortalized human meibomian gland epithelial (IHMGE) cells forming spheroids resembling meibomian gland alveoli when grown in 3D culture. Figure 7A Images of 2D and 3D spheroid cultures are depicted; brightfield images were collected using transmitted light. Figure 7B Depicted are immunofluorescence images of nuclear markers, spheroids expressing a meibomian gland acinar basal region marker (Krt5), and a merged image of the two.

[0024] Figure 8 Spheroids grown in the presence of IGF-1 E3R were significantly larger than those grown in the presence of wild-type IGF-1. On day seven of differentiation, 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.

[0025] Figure 9IGF-1 and IGF-1 E3R provide a proportional increase in lipid content in proportion to spheroid diameter. On day seven of differentiation, lipidtox intensity was measured for spheroids cultured in the presence of 0 nM, 0.1 nM, 1.6 nM, or 10 nM IGF-1 or IGF-1 E3R.

[0026] Figure 10 It is provided that IGFBP2 is more highly expressed compared to IGFBP1, IGFBP3, IGFBP4, and IGFBP6 in IHMGE spheroid cultures.

[0027] Figure 11A Describes the study design for transcriptional analysis of the effects of wild-type IGF1 treatment on IHMGE spheroids. Figure 11B Depicted are four IGFBPs that were significantly upregulated by treatment of IHMGE spheroids with wild-type IGF-1 in transcriptional analysis. Figure 11C It was depicted that IGF-1 treatment significantly upregulated genes involved in fatty acid transport, lipid synthesis, and meibomian gland formation.

[0028] Figure 12 The results show that IGFBP2 is highly expressed in the basal region of mouse meibomian glands. Figure 12 Depicted are representative immunofluorescence images of meibomian glands, IGFBP2 labeling both nuclei (DAPI, green) and proliferating cells (Ki67, red), and a merged image of the two.

[0029] Figure 13A and Figure 13B Depicts increased meibomian gland acinar proliferation in young mice compared to old mice. Cell proliferation in the meibomian glands decreases with age. Figure 13A Figure 3 depicts that the number of proliferating cells (Ki67+) in aged meibomian gland alveoli is lower than that observed in young meibomian gland alveoli. Statistical comparisons were performed using Student's t-test, n=10 per group. Figure 13B Representative immunofluorescence images of young (top) and old (bottom) meibomian glands are depicted, labeling nuclei (DAPI, green) and proliferating cells (Ki67, red). Using image analysis in ImageJ, cells within the acini that are positive for both markers (double positive) are highlighted in blue.

[0030] Figure 14A and Figure 14B Provides graphs and images showing increased proliferation in meibomian gland acini following systemic IGF-1LR3 treatment in aged mice. Figure 14AIGF-1 LR3 was shown to induce meibomian gland proliferation in aging mice. Mice were administered two doses of IGF-1 LR3 at 10 mg / kg (12 hours apart) by intraperitoneal injection, and the drug was withdrawn 24 or 48 hours after the first dose. The number of proliferating cells (Ki67+) in the meibomian gland alveoli treated with vehicle (left) or IGF-1 LR3 (right) was quantified by comparing the number of Ki67-positive cells in the meibomian gland alveoli. Statistical comparisons were performed using a two-way ANOVA followed by Tukey's multiple comparison test. N = 10 per group. Figure 14B Representative immunofluorescence images of labeled nuclei (DAPI, green) and proliferating cells (Ki67, red) are shown for meibomian glands treated with vehicle for 24 hours (top) and IGF-1LR3 for 24 hours (bottom). Cells in the acini that are positive for both markers (double positive) are highlighted in blue using image analysis in ImageJ.

[0031] Figure 15A and Figure 15B Graphs and images are provided showing that systemic IGF-1LR3 treatment in aged mice reversed atrophy and increased size in the meibomian gland region. Figure 15A Depicted are mice that were either untreated (young) or administered IGF-1LR3 at 10 mg / kg via intraperitoneal injection (5 consecutive days per week for 4 weeks) and underwent transillumination of the meibomian glands prior to sacrifice. Quantitative data from meibomian gland imaging showed an increase in gland area in IGF-1LR3-treated old mice compared to vehicle-treated control old mice. Pairwise comparisons were performed using a one-tailed Mann-Whitney test, with n = 7-10 mice per group. Figure 15B Representative images of a transilluminated meibomian gland imaging experiment are depicted, with one acinar region per image.

[0032] Figure 16A and Figure 16B Provides graphs and images showing that lipid synthesis in the meibomian glands is increased regardless of age after systemic IGF-1LR3 treatment. Figure 16A Mice were either untreated (young) or administered IGF-1LR3 at 10 mg / kg via intraperitoneal injection (5 consecutive days per week for 4 weeks) until sacrifice. Cell nuclei (DAPI, red) and meibomian gland lipids (Lipidtox, green) were labeled in eyelid sections, and lipid droplet density was quantified in ImageJ. Statistical comparisons were performed using one-way ANOVA followed by Tukey's multiple comparison test. N = 7-10 per group. Figure 16B Representative images of each group are depicted.

[0033] Figure 17Provided is a chart showing that IGF-1LR3 can be delivered systemically or by eye drops to activate the IGF1 receptor in the eyelid. Mice were administered a single dose of 10 mg / kg IGF-1LR3 by intraperitoneal injection, or treated with a vehicle (PBS) or PBS containing 5 mg / ml IGF-1LR3 applied to the ocular surface by eye drops. Animals were sacrificed 30 minutes after administration, and the eyelids were homogenized and pAKT levels were measured by pAKT ELISA as a reporter for IGF1R activation. Statistical comparisons were performed using a one-way ANOVA and subsequent Tukey's multiple comparison test. n = 3 per group.

[0034] Figure 18 IGF-1LR3 prolonged the duration of increased IGF1R signaling compared to wild-type IGF-1. The effects of wild-type IGF-1 and IGF-1LR3 on AKT S473 phosphorylation were measured 0.5 or 2 hours after administration. Statistical analysis: One-way ANOVA. n = 3 per group.

[0035] Figure 19 The present invention provides data showing that IGF-1LR3 and IGF-1Des 1-3 (which have reduced binding to the binding protein) both prolonged the duration of IGF1R activation 2 hours after administration compared to wild-type IGF-1. n = 5 per group. The effects of IGF-1, IGF-1LR3, and IGF-1Des 1-3 on AKT S473 phosphorylation were measured in the eyelids of test animals 2 hours after administration. Statistical analysis: One-way ANOVA.

[0036] Figure 20 The results provide evidence that IGF-1LR3 and IGF-1E3R (which have reduced binding to binding proteins) exhibit similar in vivo efficacy compared to IGF-1 at 1 hour post-dose. The effects of IGF-1, IGF-1LR3, and IGF-1E3R on AKT S473 phosphorylation were measured in the eyelids of test animals at 1 hour post-dose. n = 3 per group. Statistical analysis: One-way ANOVA.

[0037] Figure 21 This study provides a dose-responsive effect of IGF-1LR3 on meibomian gland proliferation in aged mice. The number of Ki67-labeled proliferating cells per 100 μm of perimeter was measured in young or aged mice after treatment with 0 mg / ml, 0.3 mg / ml, 1 mg / ml, or 3 mg / ml of IGF-1LR3 eye drops. N = 5 per group. Statistical analysis: One-way ANOVA.

[0038] Figure 22A and Figure 22BIGF-1LR3 was provided to regenerate atrophic meibomian glands in aged mice. Compared with pre-treatment and post-treatment, daily treatment with IGF-1LR3 for one month increased the area of ​​the meibomian glands. Figure 22A Depicted are individual animal glands before and after IGF-1LR3 treatment or vehicle treatment. Figure 22B Depicted are quantifications of changes in gland area following treatment with IGF-1LR3 or vehicle, assessed by transillumination meibomian gland imaging. n = 10 per group. Statistical analysis: One-way ANOVA.

[0039] Figure 23 The present study shows that IGF-1LR3 and IGF-1E3R induce similar levels of meibomian gland proliferation in aged mice after two 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 vehicle or IGF-1ER3 eye drop treatment. N = 4-5 per group. Statistical analysis: One-way ANOVA. DETAILED DESCRIPTION

[0040] The present disclosure is not limited to the aspects of the specific embodiments described in this application, which are intended to serve as separate illustrations of various aspects of the present disclosure. All various embodiments of the present disclosure will not be described herein. As will be apparent to those skilled in the art, various modifications and variations may be made to the present disclosure without departing from the spirit and scope of the present disclosure. In addition to those enumerated herein, functionally equivalent methods and devices within the scope of the present disclosure will be apparent to those skilled in the art from the foregoing description. Such modifications and variations are intended to fall within the scope of the appended claims. The present disclosure is limited only by the aspects of the full scope of the equivalents given by the appended claims and such claims.

[0041] Certain definitions

[0042] All terms are intended to be understood as meanings that can be understood by those skilled in the art. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this disclosure belongs.

[0043] The following definitions are intended to supplement those in the art and are specific to the present application and should not be attributed to any related or unrelated cases, such as any commonly owned patents or applications. Although any methods and materials similar or equivalent to those described herein can be used to test the practice of the present disclosure, preferred materials and methods are described herein. Therefore, the terms used herein are only used for the purpose of describing specific embodiments and are not intended to limit the present disclosure.

[0044] The terms used herein are for the purpose of describing a particular case only and are not intended to limit the present disclosure. In this application, unless otherwise expressly indicated, the use of the singular includes the plural. As used herein, unless the context clearly indicates otherwise, the singular forms "a," "an," and "the" are intended to include the plural.

[0045] In this application, unless otherwise indicated, the use of "or" means "and / or". As used herein, the terms "and / or" and "any combination thereof" and their grammatical equivalents are used interchangeably. These terms can express any combination specifically contemplated. For illustrative purposes only, the following phrases "A, B and / or C" or "A, B, C or any combination thereof" may mean "A alone; B alone; C alone; A and B; B and C; A and C; and A, B and C". The term "or" may be used in conjunction or disjunction unless the context clearly indicates a disjunction.

[0046] The term "about" or "approximately" means plus or minus 10% of the number to which the term refers.

[0047] As used in this specification and claims, the words "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include"), or "containing" (and any form of containing, such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method or composition of the present disclosure, and vice versa. In addition, the compositions of the present disclosure can be used to implement the methods of the present disclosure.

[0048] References in this specification to "some embodiments," "an embodiment," "one embodiment," or "other embodiments" mean that a particular feature, structure, or characteristic described in connection with these embodiments is included in at least some embodiments, but not necessarily all embodiments, of the present disclosure. To facilitate understanding of the present disclosure, certain terms and phrases are defined below.

[0049] The scope provided herein should be understood as abbreviation for all values ​​within the scope. For example, a range of 1 to 50 should be understood to include any number, combination of numbers or subranges in 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 all intermediate decimal values ​​between the above integers, such as, for example, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8 and 1.9. About subranges, " nested subranges " extending from either end of a range are particularly contemplated. For example, nested sub-ranges of the example range of 1 to 50 could include 1 to 10, 1 to 20, 1 to 30, and 1 to 40 in one direction, or 50 to 40, 50 to 30, 50 to 20, and 50 to 10 in the other direction.

[0050] The term "pharmaceutically acceptable" means approved or approvable by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia (USP) or other generally recognized pharmacopeia for use in animals, including humans.

[0051] A "pharmaceutically acceptable excipient, carrier, or diluent" refers to an excipient, carrier, or diluent that can be administered to a subject with a pharmaceutical agent without destroying its pharmacological activity and that is non-toxic when administered in a dose sufficient to deliver a therapeutic amount of the pharmaceutical agent.

[0052] 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 mammal, including, but not limited to, a human or a non-human mammal, such as a non-human primate, cow, horse, dog, sheep, or cat.

[0053] The term "optional" or "alternatively" means that the subsequent event or situation described can but need not occur, and describes the situation in which the event or situation occurs and the situation in which the event or situation does not occur. As used herein, the term "sequence identity" or sequence identity percentage (%) is the percentage of the residue in the candidate sequence that is identical with the residue in the selected sequence, after comparing sequences and introducing room as needed to realize maximum sequence identity percentage and not considering any conservative replacement as a part for sequence identity. The comparison for determining the purpose of amino acid sequence identity percentage can be achieved in many ways by those skilled 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 the appropriate parameters for measuring the comparison, including any algorithm required for realizing maximum comparison on the full length of the compared sequence.

[0054] The term "effective amount" or "therapeutically effective amount" refers to an amount of a drug sufficient to produce a beneficial or desired result. A therapeutically effective amount may vary depending on one or more of the following: the subject and disease condition being treated, the subject's weight and age, the severity of the disease condition, the mode of administration, etc., which can be readily 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. The specific dosage may vary depending on one or more of the following: the specific agent selected, the dosing regimen followed, whether it is administered in combination with other compounds, the timing of administration, the tissue to be imaged, and the physical delivery system that carries the agent. An effective amount of the active agent may be administered in a single dose or in multiple doses.

[0055] The terms "polypeptide," "oligopeptide," "peptide," and "protein," used interchangeably herein, refer to amino acid polymers of any length. The polymer may be linear or branched, it may contain modified amino acids, and it may be interrupted by non-amino acids. The term also encompasses amino acid polymers modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation to a labeling component. The definition also includes, for example, polypeptides containing one or more amino acid analogs (including, for example, unnatural amino acids, etc.), as well as other modifications known in the art.

[0056] Although certain embodiments of the present application have been shown and described herein, it will be apparent that these embodiments are provided by way of example only. Various changes, modifications, and substitutions may be made by those skilled in the art without departing from the embodiments; it will be understood that various alternatives to the embodiments described herein may be employed in practicing the methods described herein.

[0057] peptides

[0058] Meibomian glands contain meibomian cells (cells that form clusters in alveoli). When functioning normally, meibomian gland cells can proliferate, differentiate, and produce meibum, a lipid-rich secretion. Meibomian gland dysfunction can be a catalyst for the development of dry eye. In one exemplary mechanism, reduced binding of insulin-like growth factor (IGF-1) to the IGF-1 receptor (IGF1R) and resulting in reduced activation of IGF1R (or other signaling defects of IGF1R) can affect the morphology and function of the meibomian glands. For example, it can lead to meibomian gland atrophy and dysfunction, or dry eye.

[0059] The present disclosure provides polypeptides and methods for treating meibomian gland or dry eye. The polypeptide can modulate (e.g., upregulate or otherwise activate) the activity of IGF1R. The affinity of the polypeptide for at least one IGF binding protein (IGFBP) is reduced relative to wild-type IGF-1 (SEQ ID NO: 1) for the IGFBP. In one aspect, the polypeptide acts locally. The level of one or both of (1) free endogenous IGF-1 or (2) a polypeptide (an IGF-1 variant with reduced affinity for one or more IGFBPs) that binds to IGF1R can be increased after administration to a subject. Local activation of IGF1R by the polypeptide can result in a prolonged or extended pharmacodynamic effect. In another aspect, the polypeptide acts systemically. Due to the reduced affinity of the polypeptide for the IGFBP, the systemic half-life of the polypeptide is reduced.

[0060] 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, due to elevated levels of circulating IGF-1 and IGF-1 variants, IGFBPs can act to enhance systemic IGF-1 signaling. In another aspect, due to decreased local levels of free IGF-1 and IGF-1 variants, IGFBPs can act to attenuate local IGF-1 signaling. In one aspect, the one or more IGFBPs can 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 comprise IGFBP1 and IGFBP4. In some embodiments, the one or more IGFBPs comprise IGFBP2 and IGFBP3. In some embodiments, the one or more IGFBPs comprise IGFBP2 and IGFBP3. In some embodiments, the one or more IGFBPs comprise IGFBP3 and IGFBP4. In some embodiments, the one or more IGFBPs comprise IGFBP1, IGFBP2, and IGFBP3. In some embodiments, the one or more IGFBPs comprise IGFBP1, IGFBP2, and IGFBP4. In some embodiments, the one or more IGFBPs comprise IGFBP1, IGFBP3, and IGFBP4. In some embodiments, the one or more IGFBPs comprise IGFBP2, IGFBP3, and IGFBP4. In some embodiments, the one or more IGFBPs comprise IGFBP1, IGFBP2, IGFBP3, and IGFBP4.

[0061] Table 1. Peptide sequences for treating meibomian gland or dry eye

[0062]

[0063] In some embodiments, the polypeptide comprises or consists of SEQ ID NO: 1 (native human IGF-1). The polypeptide may comprise or consist of 70 amino acids. The polypeptide may strongly bind to IGF1R. The polypeptide may strongly bind to one or more IGFBPs. The affinity of native IGF-1 for IGF1R, as measured by an affinity assay, may range from about 1 nanomolar (nM) to about 10 nM.

[0064] In one aspect, the polypeptide is an IGF-1 variant. In some embodiments, the IGF-1 variant has at least 60% sequence identity with SEQ ID NO: 1. In some embodiments, the IGF-1 variant has at least 65% sequence identity with SEQ ID NO: 1. In some embodiments, the IGF-1 variant has at least 70% sequence identity with SEQ ID NO: 1. In some embodiments, the IGF-1 variant has at least 75% sequence identity with SEQ ID NO: 1. In some embodiments, the IGF-1 variant has at least 80% sequence identity with SEQ ID NO: 1. In some embodiments, the IGF-1 variant has at least 85% sequence identity with SEQ ID NO: 1. In some embodiments, the IGF-1 variant has at least 90% sequence identity with SEQ ID NO: 1. In some embodiments, the IGF-1 variant has at least 91% sequence identity with SEQ ID NO: 1. In some embodiments, the IGF-1 variant has at least 92% sequence identity with 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 can be in the range of about 1 nanomolar (nM) to about 10 nM as measured by affinity assays. In some embodiments, as measured by affinity assays with Figure 1 The polypeptide (e.g., IGF-1 variant) has an EC 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 assays described herein. 50In some embodiments, the EC of the polypeptide 50 In some embodiments, the polypeptide (e.g., IGF-1 variant) does not bind to or very weakly binds to the insulin receptor. For example, in some cases, the Kd of the interaction of the polypeptide (e.g., IGF-1 variant) is less than 1 / 10 the binding of insulin to the insulin receptor.

[0065] In some embodiments, an 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, an IGF-1 variant comprises a truncation comprising at least about 1 amino acid deletion. In some embodiments, an IGF-1 variant comprises a truncation comprising at least about 2 amino acid deletions. In some embodiments, an IGF-1 variant comprises a truncation comprising at least about 3 amino acid deletions. In some embodiments, an IGF-1 variant comprises a truncation comprising at least about 4 amino acid deletions. In some embodiments, an IGF-1 variant comprises a truncation comprising at least about 5 amino acid deletions. In some embodiments, an IGF-1 variant comprises a truncation comprising at least about 6 amino acid deletions. In some embodiments, an IGF-1 variant comprises a truncation comprising at least about 7 amino acid deletions. In some embodiments, an IGF-1 variant comprises a truncation comprising at least about 8 amino acid deletions. In some embodiments, an IGF-1 variant comprises a truncation comprising at least about 9 amino acid deletions. 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.

[0066] 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.

[0067] 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. Position numbering can be based on an alignment of the IGF-1 variant with SEQ ID NO: 1, with positions numbered from the N-terminus of SEQ ID NO: 1 to the C-terminus of SEQ ID NO: 1, starting at position 1 at the N-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 R37 deleted.

[0068] 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 3 amino acids at the N-terminus of SEQ ID NO: 1 and a deletion of the amino acid 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 a deletion of the amino acid at position 37 relative to wild-type IGF-1 (SEQ ID NO: 1).

[0069] 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 the amino acid sequence of SEQ ID NO: 3. In some embodiments, the IGF-1 variant comprises the amino acid sequence of SEQ ID NO: 4. In some embodiments, the IGF-1 variant comprises the amino acid sequence of SEQ ID NO: 5. 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 the amino acid sequence of SEQ ID NO: 7. In some embodiments, the IGF-1 variant comprises the amino acid sequence of SEQ ID NO: 8. In some embodiments, the IGF-1 variant comprises the amino acid sequence of SEQ ID NO: 12.

[0070] 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.

[0071] The polypeptide may have a reduced systemic and / or local half-life following administration to healthy adults compared to native human IGF-1. In some embodiments, the systemic and / or local half-life of the polypeptide following administration to healthy adults is reduced by at least 5% compared to native human IGF-1. In some embodiments, the systemic and / or local half-life of the polypeptide following administration to healthy adults is reduced by at least 10% compared to native human IGF-1. In some embodiments, the systemic and / or local half-life of the polypeptide following administration to healthy adults is reduced by at least 15% compared to native human IGF-1. In some embodiments, the systemic and / or local half-life of the polypeptide following administration to healthy adults is reduced by at least 20% compared to native human IGF-1. In some embodiments, the systemic and / or local half-life of the polypeptide following administration to healthy adults is reduced by at least 30% compared to native human IGF-1. In some embodiments, the systemic and / or local half-life of the polypeptide following administration to healthy adults is reduced by at least 40% compared to native human IGF-1. In some embodiments, the systemic and / or local half-life of the polypeptide is reduced by at least 50% compared to native human IGF-1 following administration to healthy adults.

[0072] The polypeptide may have reduced affinity for at least one IGFBP relative to native human IGF-1 (SEQ ID NO: 1). In some embodiments, the polypeptide has reduced affinity for IGFBP1 relative to native human IGF-1 (SEQ ID NO: 1). In some embodiments, the polypeptide has reduced affinity for IGFBP2 relative to native human IGF-1 (SEQ ID NO: 1). In some embodiments, the polypeptide has reduced affinity for IGFBP3 relative to native human IGF-1 (SEQ ID NO: 1). In some embodiments, the polypeptide has reduced affinity for IGFBP4 relative to native human IGF-1 (SEQ ID NO: 1). In some embodiments, the polypeptide has reduced affinity for IGFBP5 relative to native human IGF-1 (SEQ ID NO: 1). In some embodiments, the polypeptide has reduced affinity for IGFBP6 relative to native human IGF-1 (SEQ ID NO: 1). In some embodiments, the polypeptide has reduced affinity for IGFBP3 and IGFBP2 relative to native human IGF-1 (SEQ ID NO: 1).

[0073] 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.

[0074] IGFBPs play a key role in extending the systemic half-life of human insulin-like growth factor 1 (IGF-1). Although the unbound form of wild-type IGF-1 has a systemic half-life of 10 to 20 minutes, the binding of IGF-1 to IGFBPs increases its half-life to several hours. This extended half-life is generally considered to increase the duration of the biological effects of IGF-1. This is most evident in Laron syndrome (a dwarfism in which patients are unable to produce IGFBPs, etc.). Laron dwarf patients given high doses of systemic IGF1 do not grow significantly, in part because IGF-1 cannot persist in the circulation without the significant half-life extension provided by IGFBP binding.

[0075] Thus, it is surprising that when IGF-1 variants that escape IGFBPs are administered topically (e.g., as eye drops to treat meibomian gland dysfunction), as described herein, the opposite phenomenon is observed. For example, the desired pharmacodynamic effects of wild-type IGF-1 treatment are seen at lower levels and within a shorter timeframe relative to IGF-1 variants that are able to escape one or more IGFBPs (e.g., IGF1 LR3, IGF des1-3, IGF E3R). In other words, it is surprising that, although binding to one or more binding proteins is believed to be critical for extending the half-life and function of IGF-1, variants that escape one or more IGF-1 binding proteins (e.g., IGFBP2) can exhibit extended and / or improved pharmacodynamic effects relative to wild-type IGF-1 (SEQ ID NO: 1) when topically administered.

[0076] Cell-penetrating peptides (CPP) and skin-penetrating peptides (SPP)

[0077] The polypeptides disclosed herein may further comprise cell-penetrating peptides (CPPs) or skin-penetrating peptides (SPPs). The stratum corneum of the skin typically contains dead cells rich in keratin, which float in a lamellar lipid domain and act as a barrier to the environment. This structure can inhibit the absorption of macromolecules and transport them to the dermis and deeper. The CPPs or SPPs disclosed herein may have a high transduction efficiency, thereby enabling transdermal delivery. The CPPs or SPPs disclosed herein can change the structure of the skin barrier, allowing molecules co-formulated therewith (even if not coupled therewith) to enter and / or transfer through the skin.

[0078] The IGF-1 variants can be coupled to a cell-penetrating peptide (CPP) or skin-penetrating peptide (SPP) described herein. The CPP or SPP can 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 any one of SEQ ID NOs: 1-8, 12, or any combination thereof is coupled to the CPP or SPP. In some embodiments, a polypeptide comprising SEQ ID NO: 1 is coupled to the CPP or SPP. In some embodiments, a polypeptide comprising SEQ ID NO: 2 is coupled to the CPP or SPP. In some embodiments, a polypeptide comprising SEQ ID NO: 3 is coupled to the CPP or SPP. In some embodiments, a polypeptide comprising SEQ ID NO: 4 is coupled to the CPP or SPP. In some embodiments, a polypeptide comprising SEQ ID NO: 5 is coupled to the CPP or SPP. In some embodiments, the polypeptide comprising SEQ ID NO: 6 is coupled to a CPP or SPP. In some embodiments, the polypeptide comprising SEQ ID NO: 7 is coupled to a CPP or SPP. In some embodiments, the polypeptide comprising SEQ ID NO: 8 is coupled to a CPP or SPP. In some embodiments, the polypeptide comprising SEQ ID NO: 12 is coupled to a CPP or SPP.

[0079] Compared to compositions or formulations that are not delivered transdermally, compositions or formulations delivered transdermally can be configured to better reach steady-state levels. Compositions or formulations delivered transdermally can also bypass liver metabolism and reduce unnecessary systemic side effects, thereby improving patient compliance. The CPP or SPP disclosed herein may comprise about 5 to about 30 amino acids or consist of about 5 to about 30 amino acids. The CPP or SPP disclosed herein can allow the polypeptide to penetrate the eyelid skin, thereby allowing the polypeptide to be absorbed by the eyelid. The SPP disclosed herein may comprise a hydrophobic peptide or consist of a hydrophobic peptide. The CPP disclosed herein comprises a cationic peptide or consists of a cationic peptide. The cationic peptide may have one or more charged amino acids, such as arginine. The CPP or SPP may have low cytotoxicity to cells (e.g., human cells). The SPP disclosed herein may comprise a macromolecule transduction domain (MTD). The MTD may comprise MRAAPAVAA (SEQ ID NO: 9) or consist of MRAAPAVAA (SEQ ID NO: 9). The MTD may be derived from the membrane translocation sequence (MTS) of the Kaposi fibroblast growth factor (FGF-4) signal peptide. The CPP disclosed herein may comprise SEQ ID NO: 10 or consist of SEQ ID NO: 10. The polypeptide of SEQ ID NO: 10 can penetrate the epidermis and dermis of the skin and can be used to treat antioxidant conditions. The polypeptide of SEQ ID NO: 10 can be delivered, for example, by a transdermal spray. The CPP disclosed herein may comprise SEQ ID NO: 11 or consist of SEQ ID NO: 11. The polypeptide of SEQ ID NO: 11 can penetrate the epidermis and dermis of the skin and can be used to treat antioxidant conditions. The polypeptide of SEQ ID NO: 11 can be delivered, for example, by a transdermal spray.

[0080] Pharmaceutical composition

[0081] The present disclosure provides pharmaceutical compositions for treating meibomian gland or dry eye. The pharmaceutical compositions can be applied topically, for example, as eye drops or as a cream. The pharmaceutical compositions can be applied to the eyelids. The pharmaceutical compositions can be formulated for systemic delivery. The pharmaceutical compositions can penetrate the eyelids.

[0082] Pharmaceutical composition can include pharmaceutically acceptable carrier or adjuvant, such as, for example, hyaluronate (or hyaluronic acid), electrolyte, ophthalmic lubricant, excipient, astringent, vasoconstrictor and / or wetting agent.The example of pharmaceutically acceptable excipient can include one or more pharmaceutically acceptable excipients, and these excipients include water, saline, sucrose, lactose, malic acid, cellulose sugar, mannitol, maltitol, dextran, sorbitol, starch, agar, alginate, chitin, chitosan, pectin, tragacanth gum, gum arabic, gelatin, collagen, casein, albumin, synthetic or semisynthetic polymer or glyceride, methylcellulose, hydroxypropyl methylcellulose and polyvinylpyrrolidone.The example of electrolyte can include sodium chloride, potassium chloride, sodium bicarbonate, potassium bicarbonate, calcium chloride, magnesium chloride, trisodium citrate, hydrochloric acid, sodium hydroxide and its mixture. Pharmaceutical composition disclosed herein can include solution with one or more electrolytes. For example, in some embodiments, the electrolyte-containing solution comprises 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 cases, the molar percentage of sodium chloride is about 40% to about 60%. In some cases, the molar percentage of sodium chloride is about 40%, 45%, 50%, 55%, or 60%. In some cases, the molar percentage of potassium chloride is about 1% to about 20%. In some cases, the molar percentage of potassium chloride is about 1%, 2%, 3%, 4%, 5%, 10%, 15%, or 20%. In some cases, the molar percentage of sodium bicarbonate is about 1% to about 25%. In some cases, the molar percentage of sodium bicarbonate is about 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, or 25%. In some cases, the molar percentage of potassium bicarbonate is about 0% to about 10%. In some cases, the molar percentage of potassium bicarbonate is about 0%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%. In some cases, the molar percentage of calcium chloride is about 0% to 10%. In some cases, the molar percentage of calcium chloride is about 0%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%. In some cases, the molar percentage of magnesium chloride is about 0% to 10%. In some cases, the molar percentage of magnesium chloride is about 0%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%. In some cases, the molar percentage of trisodium citrate is about 0% to 10%. In some cases, the molar percentage of trisodium citrate is about 0%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%. In some cases, the molar percentage of hydrochloric acid is from about 0% to about 30%.In some cases, the molar percentage of hydrochloric acid is about 0%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%, 15%, 20%, 25%, or 30%. In some cases, the molar percentage of sodium hydroxide is about 0% to about 30%. In some cases, the molar percentage of sodium hydroxide is about 0%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%, 15%, 20%, 25%, or 30%.

[0083] In some embodiments, the excipient may include one or more penetration enhancers. The term "penetration enhancer" or "penetration enhancer" may refer to a compound or mixture of compounds that interacts with one or more skin components to promote drug penetration and / or passage through the skin (e.g., through the outer eyelid skin to one or more meibomian glands). Any suitable penetration enhancer known in the art may 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 their derivatives (e.g., sodium glycolate, sodium deoxycholate, etc.), fatty acids and their derivatives (e.g., oleic acid, caprylic acid, fatty acid esters (e.g., isopropyl myristate), etc.), chelating agents (e.g., EDTA, citric acid, etc.), sulfoxides (e.g., DMSO, DMA, DMF, etc.), polyols (e.g., , diethylene glycol monoethyl ether, PG, polyethylene glycol (PEG), glycerol, polyethylene glycol, etc.), alcohols (e.g., alkanols, enols, ethylene glycol, etc.), hydrocarbons (e.g., alkanes, alkenes, halogenated alkanes, squalene, squalene, mineral oil, etc.), amines, amides (e.g., cyclic amides, non-cyclic 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.

[0084] In some embodiments, pharmaceutically acceptable excipients are formulated to 1) achieve improved spreadability on the outer eyelid surface; and / or 2) avoid flowing from the outer eyelid surface to the corneal surface, thereby being suitable for transdermal delivery through the outer eyelid. In some embodiments, pharmaceutically acceptable excipients include improved spreadability and rheological characteristics, resulting in easier application and spreading to the eyelid surface, and will not flow at the subject's body temperature, especially after being applied to the subject's skin. In some embodiments, the pharmaceutical compositions of the present disclosure are formulated so that the cohesiveness of the preparation does not change significantly after being applied to the subject's skin (e.g., eyelid). Examples of suitable additives that impart appropriate cohesiveness to the preparation can include, for example, additives that increase the viscosity of the preparation, such as waxes, paraffin waxes, and elastomers. In some embodiments, the viscosity of the preparation does not change significantly when heated from room temperature to a temperature closer to the subject's body temperature.

[0085] In some embodiments, the pharmaceutically acceptable preparation is an ointment comprising an ointment base miscible with water. In some embodiments, the pharmaceutically acceptable preparation is an ointment comprising a paraffin ointment base. In some embodiments, the ointment comprises one or more of white soft paraffin, 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 soft paraffin, mineral oil, propylene glycol, ST cyclomethicone -5NF, labrasol, propylene carbonate, steareth -2, ST emulsifier 10 and ST elastomer -10.

[0086] 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 soft paraffin / 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 soft paraffin / 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 soft paraffin / petrolatum, mineral oil, propylene glycol, cyclomethicone, emulsifier 10, ST-Elastomer-10, methylparaben, anhydrous disodium hydrogen phosphate, anhydrous citric acid, propylparaben, and purified water.

[0087] The pharmaceutical compositions disclosed herein can be administered to a subject. A therapeutically effective amount of the pharmaceutical compositions disclosed herein can be administered to a subject. The pharmaceutical compositions can be administered via any of the delivery modes disclosed herein, such as eye drops or creams. Eye drops can be administered via a dropper. The pharmaceutical compositions can be administered to the subject's eye, or a portion thereof. The pharmaceutical compositions can be administered to the eyelid, such as the outer eyelid.

[0088] The subject may have meibomian gland dysfunction. Meibomian gland dysfunction can lead to altered tear film composition, ocular surface disease, eye and eyelid discomfort, and evaporative dry eye. Symptoms of meibomian gland dysfunction include dryness, burning, itching, redness, crusting discharge, increased tearing, blurred vision, and light sensitivity.

[0089] In some embodiments, compared with the object that does not receive the pharmaceutical composition, the pharmaceutical composition disclosed herein is applied to the object and can cause the size of the meibomian gland to increase. In some embodiments, compared with the object that does not receive the pharmaceutical composition, the pharmaceutical composition disclosed herein is applied to the object and can cause the meibomian gland atrophy to reduce. In some embodiments, compared with the object that does not receive the pharmaceutical composition, the pharmaceutical composition disclosed herein is applied to the object and can cause the meibomian gland to regenerate. In some embodiments, compared with the object that does not receive the pharmaceutical composition, the pharmaceutical composition disclosed herein is applied to the object and can cause the age-related meibomian gland atrophy to reverse. In some embodiments, compared with the object that does not receive the pharmaceutical composition, the pharmaceutical composition disclosed herein is applied to the object and can cause the function of one or more meibomian gland cells to increase. For example, in some cases, the function increase can include an increase in Akt phosphorylation (or an increase in the duration of activity of Akt phosphorylation), an increase in the phosphorylation of IGF1R itself, or an increase in the phosphorylation of other downstream signal transduction molecules that are phosphorylated when IGF1R is activated. In some embodiments, compared with the object that does not receive the pharmaceutical composition, the pharmaceutical composition disclosed herein is applied to the object and can cause corneal proliferation and / or repair to increase. In some embodiments, compared to an object that has not received the pharmaceutical composition, applying a pharmaceutical composition disclosed herein to an object can result in increased corneal healing. In some embodiments, compared to an object that has not received the pharmaceutical composition, applying a pharmaceutical composition disclosed herein to an object can result in increased activation of IGF1 receptors (IGF1R) in the meibomian glands. In some embodiments, compared to an object that has not received the pharmaceutical composition, applying a pharmaceutical composition disclosed herein to an object can result in an increase in the duration of IGF1R activation in the meibomian glands. In some embodiments, compared to an object that has not received the pharmaceutical composition, applying a pharmaceutical composition disclosed herein to an object can result in an increase in the lipid content of the meibomian glands.

[0090] Administration of the pharmaceutical composition to a subject with meibomian gland dysfunction can result in a median increase in the surface area or volume of the meibomian glands of the subject, compared to a subject that did not receive the pharmaceutical composition. In some embodiments, the surface area or volume of the meibomian glands of the inner eyelid surface of the subject increases by at least 5%, compared to a subject that did not receive the pharmaceutical composition. In some embodiments, the surface area or volume of the meibomian glands of the subject increases by at least 6%, compared to a subject that did not receive the pharmaceutical composition. In some embodiments, the surface area or volume of the meibomian glands of the subject increases by at least 7%, compared to a subject that did not receive the pharmaceutical composition. In some embodiments, the surface area or volume of the meibomian glands of the subject increases by at least 8%, compared to a subject that did not receive the pharmaceutical composition. In some embodiments, the surface area or volume of the meibomian glands of the subject increases by at least 9%, compared to a subject that did not receive the pharmaceutical composition. In some embodiments, the surface area or volume of the meibomian glands of the subject increases by at least 10%, compared to a subject that did not receive the pharmaceutical composition. In some embodiments, the surface area or volume of the meibomian glands of the subject increases by at least 15%, compared to a subject that did not receive the pharmaceutical composition. In some embodiments, the surface area or volume of the meibomian glands of the subject increases by at least 20%, compared to a subject that did not receive the pharmaceutical composition. In some embodiments, the surface area or volume of the meibomian glands of a subject is increased by at least 30% compared to a subject that did not receive the pharmaceutical composition. In some embodiments, the surface area or volume of the meibomian glands of a subject is increased by at least 40% compared to a subject that did not receive the pharmaceutical composition. In some embodiments, the surface area or volume of the meibomian glands of a subject is increased by at least 50% compared to a subject that did not receive the pharmaceutical composition. In some embodiments, the surface area or volume of the meibomian glands of a subject is increased by at least 60% compared to a subject that did not receive the pharmaceutical composition.

[0091] Administration of the pharmaceutical composition to subjects with meibomian gland dysfunction can result in a median increase in lipid content within the meibomian glands, compared to subjects who did not receive the pharmaceutical composition. In some embodiments, the lipid content within the meibomian glands increases by at least 5%, compared to subjects who did not receive the pharmaceutical composition. In some embodiments, the lipid content within the meibomian glands increases by at least 6%, compared to subjects who did not receive the pharmaceutical composition. In some embodiments, the lipid content within the meibomian glands increases by at least 7%, compared to subjects who did not receive the pharmaceutical composition. In some embodiments, the lipid content within the meibomian glands increases by at least 8%, compared to subjects who did not receive the pharmaceutical composition. In some embodiments, the lipid content within the meibomian glands increases by at least 9%, compared to subjects who did not receive the pharmaceutical composition. In some embodiments, the lipid content within the meibomian glands increases by at least 10%, compared to subjects who did not receive the pharmaceutical composition. In some embodiments, the lipid content within the meibomian glands increases by at least 15%, compared to subjects who did not receive the pharmaceutical composition. In some embodiments, the lipid content within the meibomian glands increases by at least 20%, compared to subjects who did not receive the pharmaceutical composition. In some embodiments, the lipid content within the meibomian glands increases by at least 30%, compared to subjects who did not receive the pharmaceutical composition. In some embodiments, the lipid content of the meibomian glands is increased by at least 40% compared to a subject that did not receive the pharmaceutical composition. In some embodiments, the lipid content of the meibomian glands is increased by at least 50% compared to a subject that did not receive the pharmaceutical composition. In some embodiments, the lipid content of the meibomian glands is increased by at least 60% compared to a subject that did not receive the pharmaceutical composition.

[0092] Compared to the object that does not receive the pharmaceutical composition, the pharmaceutical composition is applied to the object suffering from meibomian gland dysfunction and the median of the lipid mass in the meibomian glands can be increased. Lipid mass can be assessed by having a lower melting point (e.g., waxy degree) than the lipid (e.g., meibum) of the object that does not receive the pharmaceutical composition. In other words, the lipid (e.g., meibum) with a lower average melting point can have improved quality relative to the lipid (e.g., meibum) with a higher average melting point. In some embodiments, the lipid mass in the meibomian glands is increased by at least 5% compared to the object that does not receive the pharmaceutical composition. In some embodiments, the lipid mass in the meibomian glands is increased by at least 6% compared to the object that does not receive the pharmaceutical composition. In some embodiments, the lipid mass in the meibomian glands is increased by at least 7% compared to the object that does not receive the pharmaceutical composition. In some embodiments, the lipid mass in the meibomian glands is increased by at least 8% compared to the object that does not receive the pharmaceutical composition. In some embodiments, the lipid mass in the meibomian glands is increased by at least 9% compared to the object that does not receive the pharmaceutical composition. In some embodiments, the lipid mass in the meibomian glands is increased by at least 10% compared to the object that does not receive the pharmaceutical composition. In some embodiments, the lipid mass within the meibomian glands is increased by at least 15% compared to a subject that did not receive the pharmaceutical composition. In some embodiments, the lipid mass within the meibomian glands is increased by at least 20% compared to a subject that did not receive the pharmaceutical composition. In some embodiments, the lipid mass within the meibomian glands is increased by at least 30% compared to a subject that did not receive the pharmaceutical composition. In some embodiments, the lipid mass within the meibomian glands is increased by at least 40% compared to a subject that did not receive the pharmaceutical composition. In some embodiments, the lipid mass within the meibomian glands is increased by at least 50% compared to a subject that did not receive the pharmaceutical composition. In some embodiments, the lipid mass within the meibomian glands is increased by at least 60% compared to a subject that did not receive the pharmaceutical composition.

[0093] Administration of the pharmaceutical composition to a subject with meibomian gland dysfunction can result in an increase in lipid release from the alveoli of the meibomian glands, compared to a subject that has not received the pharmaceutical composition. In some embodiments, lipid release from the alveoli of the meibomian glands increases by at least 5%, compared to a subject that has not received the pharmaceutical composition. In some embodiments, lipid release from the alveoli of the meibomian glands increases by at least 10%, compared to a subject that has not received the pharmaceutical composition. In some embodiments, lipid release from the alveoli of the meibomian glands increases by at least 15%, compared to a subject that has not received the pharmaceutical composition. In some embodiments, lipid release from the alveoli of the meibomian glands increases by at least 20%, compared to a subject that has not received the pharmaceutical composition. In some embodiments, lipid release from the alveoli of the meibomian glands increases by at least 30%, compared to a subject that has not received the pharmaceutical composition. In some embodiments, lipid release from the alveoli of the meibomian glands increases by at least 40%, compared to a subject that has not received the pharmaceutical composition. In some embodiments, lipid release from the alveoli of the meibomian glands increases by at least 50%, compared to a subject that has not received the pharmaceutical composition. In some embodiments, lipid release from the alveoli of the meibomian glands increases by at least 75%, compared to a subject that has not received the pharmaceutical composition. In some embodiments, lipid release from the alveoli of the meibomian glands is increased by at least 100% compared to a subject that did not receive the pharmaceutical composition. In some embodiments, lipid release from the alveoli of the meibomian glands is increased by at least 150% compared to a subject that did not receive the pharmaceutical composition. In some embodiments, lipid release from the alveoli of the meibomian glands is increased by at least 200% compared to a subject that did not receive the pharmaceutical composition. In some embodiments, lipid release from the alveoli of the meibomian glands is increased by at least 300% compared to a subject that did not receive the pharmaceutical composition.

[0094] Administration of a pharmaceutical composition to a subject with meibomian gland dysfunction can result in an increase in one or more pharmacodynamic effects, such as an increase in the duration of Akt phosphorylation in meibomian gland cells compared to a subject that did not receive the pharmaceutical composition. In some embodiments, the duration of Akt phosphorylation in meibomian gland cells is increased by at least 5% compared to a subject that did not receive the pharmaceutical composition. In some embodiments, the duration of Akt phosphorylation in meibomian gland cells is increased by at least 10% compared to a subject that did not receive the pharmaceutical composition. In some embodiments, the duration of Akt phosphorylation in meibomian gland cells is increased by at least 15% compared to a subject that did not receive the pharmaceutical composition. In some embodiments, the duration of Akt phosphorylation in meibomian gland cells is increased by at least 20% compared to a subject that did not receive the pharmaceutical composition. In some embodiments, the duration of Akt phosphorylation in meibomian gland cells is increased by at least 30% compared to a subject that did not receive the pharmaceutical composition. In some embodiments, the duration of Akt phosphorylation in meibomian gland cells is increased by at least 40% compared to a subject that did not receive the pharmaceutical composition. In some embodiments, the duration of Akt phosphorylation in meibomian gland cells is increased by at least 50% compared to a subject that did not receive the pharmaceutical composition. In another embodiment, the change in one or more pharmacodynamic effects can be increased phosphorylation of any downstream target resulting from IGF1R activation.

[0095] Pharmaceutical compositions 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 relative to wild-type IGF-1 (SEQ ID NO: 1).

[0096] In some embodiments, the pharmaceutical composition does not comprise any other phospholipid deposition inducing agent. In some embodiments, the pharmaceutical composition does not comprise either azithromycin or doxycycline.

[0097] Methods of treatment, administration and use

[0098] 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 eye condition in a subject in need thereof. In some aspects, the methods comprise administering a pharmaceutical composition as provided herein. In some aspects, the methods comprise administering a polypeptide as provided herein. In some aspects, the methods comprise administering an IGF-1 variant as 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 the pharmaceutical composition as provided herein is administered.

[0099] Also provided herein are uses of the pharmaceutical compositions provided herein for treating eye disorders.

[0100] The subject may have a disease or condition that is in need of treatment provided by the present disclosure. The disease or condition may include dry eye, meibomian gland dysfunction, and / or Sjögren's syndrome.

[0101] For treatment, the amount of the pharmaceutical composition provided herein is an amount effective to produce the desired effect, e.g., to treat or alleviate the effects and / or symptoms of an eye disorder in a subject in need thereof. An effective amount can be provided in one or a series of administrations of the pharmaceutical composition provided herein.

[0102] A subject suffering from an ocular disorder can be identified by any diagnostic or prognostic assay known in the art, or a combination thereof.

[0103] The method for treating a subject in need thereof can further comprise administering to the subject, sequentially, separately, or simultaneously, at least one additional therapy, such as artificial tears or punctal plugs.

[0104] In any case, the multiple therapeutic agents can be administered in any order, or even simultaneously. If administered simultaneously, the multiple therapeutic agents can be provided in a single unified form or in multiple forms (by way of example only, as a single pill or as two separate pills). One of the therapeutic agents can be administered in multiple doses, or both therapeutic agents can be administered in multiple doses. If not administered simultaneously, the time intervals between multiple doses can vary from more than zero weeks to less than four weeks. In addition, the combined methods, compositions, and formulations are not limited to the use of only two agents.

[0105] Reagent test kit

[0106] The present disclosure provides a kit comprising a pharmaceutical composition disclosed herein and an eye dropper. The eye dropper can be configured to deliver the pharmaceutical composition as an eye drop solution.

[0107] Example

[0108] Example 1: In vitro efficacy of wild-type IGF-1 and IGF-1 variants in the presence and absence of IGFBPs

[0109] Materials and methods

[0110] In vitro potency assay of DU145 cells

[0111] 15 x 10 6 DU145 cells (ATCC #HTB-81) were reseeded and expanded in T175 culture flasks containing DMEM / F12 (1:1) medium (Gibco 11320-033) supplemented with 10% FBS (Gibco 10437-028) and 10 μg / mL gentamicin (Gibco 15710-064) ("culture medium"). At 80%-90% confluence, cells were trypsinized, counted manually using a hemocytometer, and cultured at 3×10 5 The cells were resuspended in culture medium at 150,000 cells / mL and then reseeded in 24-well plates (Corning 3524) in 0.5 mL aliquots / well (150,000 cells / well). 14-16 hours after reseeding, the culture medium was removed, and the cells were washed with 1 mL of PBS and cultured in 225 μL of serum-free culture medium supplemented with 0.2% BSA (Millipore #A3059) for 6 hours. 25 μL aliquots of the following growth factor dilution series were added to the cells in duplicate to give final concentrations in the wells: 457, 137, 41, 12.5, 3.7, 1.11, and 0.33 nM IGF-1 (SEQ ID NO: 1), IGF-1Ea (SEQ ID NO: 7), IGF-1Des1-3 R37X (SEQ ID NO: 6), IGF-1Des1-3 (SEQ ID NO: 8), and IGF-1E3R (SEQ ID NO: 4). After incubation at 37°C for 15 minutes, the medium was aspirated and the cells were lysed in 150 μL of 1xPTR buffer (Extraction Buffer 5xPTR, Abcam ab193970) containing 1x Extraction Enhancer Buffer (Extraction Enhancer Buffer 50x, Abcam ab193971) and protease / phosphatase inhibitors (one Pierce microplate [#A32959] / 10 mL 1xPTR). After incubation on ice for 15-30 minutes, the plate was vortexed and the lysate was transferred to an Eppendorf tube and stored at -80°C to assess the IGF1R-AKT signaling intensity using the human / mouse / rat phosphorylated Akt (S473) pan-specific DuoSetIC ELISA (RnD#DYC887B).

[0112] Incubation of IGFBP and IGF-1 in DU145 cells

[0113] To determine the in vitro efficacy 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), rhIGF1LR3 (SEQ ID NO: 12), or rhIGF1 E3R (SEQ ID NO: 4) at a 1:1, 2:1, or 4:1 molar ratio. After incubation for 1 hour at room temperature, the complexes containing the IGF1 variants were added to the cells in triplicate at a final concentration of 25 nM.

[0114] result

[0115] The dose-dependent effects of IGF-1 (SEQ ID NO: 1), IGF-1Ea (SEQ ID NO: 7), IGF-1Des1-3R37X (SEQ ID NO: 6), IGF-1Des1-3 (SEQ ID NO: 8), and IGF-1E3R (SEQ ID NO: 4) on AKT S473 phosphorylation were determined in DU145 cells. 50 The value is 6.0 nM. The EC of IGF-1Ea 50 The EC value of IGF-1Des 1-3R37X is 8.7 nM. 50 The EC value of IGF-1Des 1-3 is 4.1 nM. 50 The EC value of IGF-1E3R is 2.3 nM. 50 The value is 4.5nM, such as Figure 1 This indicates that wild-type IGF-1 and the tested IGF-1 variants have similar EC 50 value.

[0116] To determine the differences in the ability of various IGF1 mutants to bind to IGFBPs, a literature search was performed. Figure 2As shown. In L6 rat myoblast conditioned medium containing various binding proteins, competition assays reported that the affinity of BP for IGF-1LR3 (SEQ ID NO: 12), LG3 (long IGF-1E3G), long IGF-1, and IGF-1Des 1-3 (SEQ ID NO: 3) was reduced by 1 / 690, 1 / 112, 1 / 5.5, and 1 / 38, respectively (see Francis, GL et al., 8 (3) J. Mol. Endocrinol. 213-223, 1992). In another study using bovine IGFBP2, it was reported that the binding affinity of IGF-1E3R (SEQ ID NO: 4) and IGF E3G for IGFBP2 was reduced by 1 / 230 and 1 / 59, respectively (see King, R. et al., 8 J. Mol. Endocrinol. 29-41, 1992). This suggests that certain IGF-1 variants, such as IGF-1 Des 1-3 (SEQ ID NO: 3), IGF-1 E3R (SEQ ID NO: 4), and IGF-1 LR3 (SEQ ID NO: 12), have reduced affinity for IGFBPs.

[0117] To determine 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-1LR3 (SEQ ID NO: 12), or IGF-1E3R (SEQ ID NO: 4) at a ratio of IGF-1 protein to IGFBP of 1:1, 1:2, or 1:4. Figure 3A and Figure 3B It was shown that IGF-1LR3 (SEQ ID NO: 12) and IGF-1E3R (SEQ ID NO: 4) were less inhibited by IGFBP2 and IGFBP3 than wild-type IGF-1 (SEQ ID NO: 1).

[0118] Example 2: Wild-type IGF-1 promotes the growth of spheroids.

[0119] Materials and methods

[0120] In vitro potency assay (pAKT assay)

[0121] 5 x 10 6Immortalized human meibomian gland epithelial cells (IHMGEC: ATCC #CRL-3472) were reseeded and expanded in T75 culture flasks containing KSFM medium (Gibco: #10724-011) supplemented with 5 μg / l human recombinant EGF (Gibco #10450-013) and bovine pituitary extract (BPE) (Gibco #13028-014), MEMNEAA (Gibco #11140-050) and penicillin-streptomycin (Gibco #15140-122) ("Proliferation Medium"). At 90%-95% confluence, cells were trypsinized and counted using an EVE automated cell counter from NanoEntek at 4×10 5Cells were resuspended in culture medium at 100 cells / ml and then re-seeded in 12-well plates (ThermoFisher #FB012928) in 1 ml aliquots / well. 36-42 hours after reseeding, the culture medium was removed and the cells were washed twice with 1 ml PBS before being cultured for 6 hours in 450 μl DMEM / F12 (1:1) medium (Gibco: #11320-033) supplemented with penicillin-streptomycin (Pen Strep) (Gibco #15140-122) and 0.2% BSA (Millipore #A3059) ("serum starvation medium"). 50 μl aliquots of the following rh-IGF-1 (SEQ ID NO: 1) dilution series were added to the cells in duplicate: 10,000, 1,000, 100, 10, 1, 0.1, 0 nM. After incubation at 37°C for 15 minutes, the culture medium was aspirated and the cells were lysed in 100 μl of M-PER (Thermo Scientific #78501) containing protease / phosphatase inhibitors (one Pierce microplate [#A32961] / 10 ml M-PER). After incubation on ice for 15-30 minutes, the plate was vortexed and the lysate was transferred to a microcentrifuge tube and stored at -80°C to assess the intensity of IGFR1-AKT signaling using the phosphorylated Akt (S473) pan-specific DuoSetIC ELISA (RnD #DYC887B-2). The ELISA was performed in a 384-well plate according to the manufacturer's recommendations with slight modifications. Briefly, the wells of a 384-well plate were coated with 25 μl of 6 μg / ml PBS-containing phosphorylated Akt1 (S473) capture antibody (#841692) (25 μl / 384 well), sealed, and left at room temperature overnight. 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 at room temperature for 1-2 hours. 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 at room temperature for 2 hours. The 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 at room temperature for 1 hour. Wells were washed four times with 100 μl of PBS containing 0.05% Tween 20 and incubated with 25 μl of streptavidin-HRP A (RnD#890803) diluted 1:200 in PBS containing 1% BSA for 20 min 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 TMB substrate solution (Abcam TMB ELISA Substrate High Sensitivity (ab171523; Lot No. GR3427893-1)) and incubated at room temperature for 10-20 min. 12.5 μl of stop solution (RnD# 895926 from auxiliary kit 2) was added and BMG Labtech was used. Plus microplate reader to measure the OD of each well 450 EC was calculated using Prism 9. 50 value.

[0122] In vitro human meibomian gland epithelial cell growth assay

[0123] Live cell imaging assay. 5 x 10 6 Immortalized human meibomian gland epithelial cells (IHMGEC: ATCC #CRL-3472) were reseeded in T75 culture flasks containing proliferation medium and expanded. At 90%-95% confluence, cells were trypsinized and counted using an EVE automated cell counter from NanoEntek. 5 Cells were resuspended in proliferation medium at 100 cells / ml and then re-seeded in two 12-well plates (Thermo Fisher #FB012928) in 1 ml aliquots / well. After 36 hours of re-seeding, the proliferation medium was removed and washed twice with 1 ml PBS and cultured in 500 μl serum starvation medium supplemented with 0, 0.1, 1, 10, 100 or 1000 nM human LR3-IGF-1 (PeproTech: #100-11R3) (4 independent wells for each condition). Every 24 hours, cells in the center of each well were imaged using an Eclipse Ti microscope (Nikon) with a 10X PlanFluor Ph1 lens (Nikon) and an iXon Life888EMCCD camera.

[0124] Cell Titer-Glo Luminescent Cell Viability Assay. 5 x 10 6 Immortalized human meibomian gland epithelial cells (IHMGEC: ATCC #CRL-3472) were reseeded in T75 culture flasks containing proliferation medium and expanded. At 90%-95% confluence, cells were trypsinized and counted using an EVE automated cell counter from NanoEntek at 1 x 10 5Cells were resuspended in proliferation medium at 100 cells / ml and then reseeded in two 12-well plates (ThermoFisher FB012928) in 1 ml aliquots / well. 36 hours after reseeding, the proliferation medium was removed and the cells were washed twice with 1 ml PBS and cultured in 500 μl serum starvation medium supplemented with 0, 0.1, 1, 10, 100, or 1000 nM human IGF-1 (R&D 291-G1). 58 hours later, the medium in each well was replaced with 200 μl DMEM / F12 and an equal volume of CellTiter- After 10 minutes on an orbital shaker, two 100 μl aliquots from each 12 well were transferred to a 96-well flat-bottom black fluorescent tracking plate (Greiner #655076) and plated using a BMG Labtech Luminescence was measured at 560-580 nm using a ELISA Plus microplate reader. As a measure of IGF1-mediated cell proliferation, the RLU values ​​of IGF1-treated IHMGEC were normalized to those of IHMGEC cultured in the absence of human IGF1.

[0125] result

[0126] Figure 4 The dose-dependent effect of IGF-1 on AKT S473 phosphorylation in IHMGECs is depicted. 50 The value is approximately 0.07 nanomolar (nM). Figure 5 Depicted are the results of a live cell imaging assay: upon IGF-1 stimulation, IHMGECs spread and proliferate at an increasing rate in a dose-dependent manner. Figure 6 Depicted are the results of human IGF-1 stimulation in IGHMECs following a cell viability assay: cells proliferated in a dose-dependent manner in response to IGF-1.

[0127] Example 3: IGF1 E3R promotes spheroid growth more than wild-type IGF-1.

[0128] Materials and methods

[0129] 3D culture of IHGMGE cells

[0130] 2500 immortalized human meibomian gland epithelial cells (IHMGEC, ATCC, #CRL-3472) were seeded in 50 μL drops of matrix (Matrigel, Corning #354230) per well of a 24-well plate, and each well of the 24-well plate was covered with proliferation medium, which was 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% penicillin / streptomycin (Gibco, #15140-122). Spheroids were grown for 8 days in proliferation medium and then in the differentiation phase for 7 days in the presence of differentiation medium supplemented with 10 μg / mL gentamicin (Gibco, #15710-064), 1% penicillin / streptomycin (Gibco, #15140-122), 20 μM rosiglitazone (Sigma Aldrich, #R2408), plus or minus various amounts of recombinant human insulin-like growth factor-1 (rhIGF1, PeproTech #100-11) or IGF1-E3R (SEQ ID NO: 12) (DMEM:F12 medium (Gibco #11320-033). N = 2 per condition.

[0131] On the sixth day of differentiation, spheroids were fixed in 4% PFA and processed for immunofluorescence using an anti-Krt5 antibody (purified anti-keratin 5 polyclonal chicken antibody, Biolegend #905903) and detected with an AF594-labeled anti-chicken secondary antibody (goat anti-chicken IgY (H+L) secondary antibody, Alexa Fluor 594, Thermo #A-11042). For images of 2D and 3D spheroid cultures, bright field images were collected using transmitted light.

[0132] Measurement of spheroid growth of IHGMGE cells

[0133] IHMGE cells were cultured as described above. To measure proliferation, the diameter of each spheroid was measured on day 7 of differentiation using ImageJ's Fiji 2. To do this, 25 bright-field z-images were captured, spanning a total of 1 mm, and a single final image was generated using maximum image projection.

[0134] Measurement of lipid production in spheroids

[0135] IHMGE cells were cultured as described above. On day 6 of differentiation, 1:3000 LipidTOX Green neutral lipid dye (Invitrogen, #H34475) was added to the wells. To measure lipid production, 25 4x images in the z-axis direction, spanning 1 mm, were taken on day 7 of differentiation using a fluorescence microscope with a filter set suitable for AlexaFluor 488 dye or fluorescein. After maximum image projection, the average signal per spheroid was calculated using QuPath software (https: / / qupath.github.io / ).

[0136] Quantification of IGFBPs in spheroids

[0137] IHMGE cells were cultured as described above. On the sixth day of differentiation, duplicate cells were merged, lysed in 200 μl 2x extraction buffer (5X PTR, Abcam#, ab193970), and fully vortexed. The lysate was then frozen at -80 degrees Celsius. During analysis, according to the manufacturer's instructions, each sample was quantified using Pierce detergent-compatible Bradford assay kit (Thermo, #23246), and analyzed by human IGF signal transduction array C1 (Ray Biotech, #AAH-IGF-1-4).

[0138] result

[0139] like Figure 7A and Figure 7B As shown in Figure 2, under conditions that promote differentiation, IHMGE cells grown in 3D culture developed into spheroid structures that resembled meibomian gland acini. Figure 7A and Figure 7B As shown, spheroids expressed markers of the basal zone of the meibomian gland acini, including Krt5, and exhibited proliferation in the basal zone.

[0140] On day six of differentiation, 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. Figure 8 The results show that spheroids grown in the presence of IGF-1E3R are significantly larger than those grown in the presence of wild-type IGF-1. In addition, 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-1E3R was measured on day 7 of differentiation. IGF-1 and IGF-1E3R proportionally increase the lipid content in spheroids, as shown in Figure 2. Figure 9 Cells were lysed for quantification of IGFBPs. Figure 10It is provided that IGFBP2 is more highly expressed compared to IGFBP1, IGFBP3, and IGFBP4 in IHMGE spheroid cultures.

[0141] Example 4: IGF1 treatment upregulates genes involved in fatty acid transport, lipid synthesis, and meibomian gland formation

[0142] Materials and methods

[0143] 2500 immortalized human meibomian gland epithelial cells (IHMGEC, ATCC, #CRL-3472) were seeded in 50 μL drops of matrix (Geltrex, ThermoFisher #A1413201) per well of a 24-well plate covered with proliferation medium, which was 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% penicillin / streptomycin (Gibco, #15140-122). Spheroids were grown in proliferation medium for 14 days and then in the differentiation stage for 4 days in the presence of differentiation medium, which was DMEM:F12 medium (Gibco #11320-033) supplemented with 10 μg / mL gentamicin (Gibco, #15710-064), 1% penicillin / streptomycin (Gibco, #15140-122), 20 μM rosiglitazone (Sigma Aldrich, #R2408), plus or minus 100 nM recombinant human insulin-like growth factor-1 (rhIGF1, PeproTech #100-11).

[0144] According to the manufacturer's instructions, RNA extraction from spheroid cultures differentiated for 4 days was performed by TRIzol extraction (Invitrogen #15596026), followed by RNA cleaning and concentration kit 5 (RNA Clean & Concentrator-5, Zymo Research #R1013) for RNA purification steps. In short, n = 4 samples under each condition. The matrix droplet containing the spheroids was transferred to a microcentrifuge tube, and 500 μL TRIzol was added to lyse the cells, followed by 100 μL chloroform, the aqueous phase was transferred to an RNase-free tube, and mixed with an equal volume of 100% ethanol. The mixture was purified using a Zymo-Spin IC column, including DNA enzyme I treatment, all according to the protocol. Before the total RNA sample was submitted to Novogene, RNA quality and quantity were assessed with nanodrop (A260 / A280=1.8-2, A260 / A230>=1.8). At Novogene, RNA purity and integrity were confirmed. Then, a Poly A-enriched mRNA library was prepared and its quality was subsequently checked (library QC). Next, the library was sequenced using the Illumina NovaSeq PE150 platform using a 150bp paired-end sequencing strategy (6G raw data per sample), and the quality of the resulting data was also checked (data QC).

[0145] Fastq files of paired-end RNA sequencing reads were aligned to the reference genome hg38 using STAR v.2.6.0a (Dobin, A. et al., 29(1) Bioinformatics 15–21, 2013) (Schneider, VA et al., 27(5) Genomeresearch 849–864, 2017). Gene-level counts of 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 < 10, differential expression analysis was performed using the R package DESeq2 v.1.40.2 (Love, MI et al., 15(12) Genomebiology 550, 2014). Gene set enrichment analysis (GSEA) (Subramanian, A. et al., 102(43) Proc Natl Acad Sci US A. 15545-15550, 2005) was performed to detect enriched pathways for human gene sets from MSigDB (Liberzon A. et al., 27(12) Bioinformatics 1739-1740, 2011). Overrepresentation analysis was performed using Fisher's exact test on a gene set related to meibomian gland formation collected from the literature, with selected upregulated (log2 fold change > 1, adjusted p-value < 0.25) and downregulated (log2 fold change < -1, adjusted p-value < 0.25) genes. Heat maps were generated using Morpheus (Broad Institute).

[0146] result

[0147] 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 plus IGF1. RNA sequencing was used to determine the effects of wild-type IGF1 on gene expression in IHMGE spheroids. Figure 11A Treatment of IHMGE spheroids with wild-type IGF1 significantly upregulated four IGFBPs, namely IGFBP2, IGFBP5, IGFBP4, and IGFBP6. Figure 11B Among them, IGFBP2 is the most significantly upregulated IGFBP in response to wild-type IGF1 treatment, as shown in Figure 11B Treatment of IHMGE spheroids with wtIGF1 significantly upregulated genes involved in fatty acid transport, lipid synthesis, and meibomian gland formation. Figure 11C The gene panel of interest was adapted from Butovich I., 163 Exp Eye Res. 2-16, 2017.

[0148] Example 5: In vivo effects of IGF-1 variants on mouse meibomian glands

[0149] Materials and methods

[0150] Animal studies

[0151] Wild-type (WT) female C57BL / 6J mice (Jackson Labs, strain #000664) were purchased at 2 months or 1-1.5 years old. All animal studies were performed in accordance with the relevant protocols.

[0152] Drug administration

[0153] For dosing studies, mice were administered vehicle (endotoxin-free PBS, EMD Millipore, TMS-012-A) or 10 mg / kg (mg / kg) IGF-1LR3 (SEQ ID NO: 12) suspended in vehicle. Mice were dosed using an insulin syringe (BD, 329424). Mice were given two intraperitoneal (IP) injections of IGF-1LR3 (12 hours apart) and sacrificed 24 or 48 hours after dosing as described below (see results). Figure 14A and 14B Mice were administered vehicle or 10 mg / kg IGF-1LR3 by intraperitoneal injection daily for 5 days, followed by a 2-day rest period for 4 weeks and then sacrificed as described below (see results). Figure 15A 、 15B Animals were given a single intraperitoneal injection of 10 mg / kg IGF-1LR3 or 10 μl of an IGF-1LR3 vehicle at a concentration of 5 mg / ml and then sacrificed as described below (for results, see Figure 17 ).

[0154] exist Figure 18 Animals were administered 10 μl of eye drops containing 1 mg / ml of wild-type IGF-1 (SEQ ID NO: 1) or 1.27 mg / ml of IGF-1LR3 (SEQ ID NO: 12) at equimolar concentrations based on molecular weight and then sacrificed as previously described 0.5 or 2 hours after administration. Eyelids were removed and frozen on dry ice and then stored at -80°C until P-AKT ELISA analysis was performed.

[0155] exist Figure 19Animals were administered 10 μl of eye drops containing 1 mg / ml wild-type IGF-1 (SEQ ID NO: 1), 1.27 mg / ml IGF-1LR3 (SEQ ID NO: 12), or 1 mg / ml rhIGF-1Des1-3 (SEQ ID NO: 3) at equimolar concentrations based on molecular weight and then sacrificed as previously described 2 hours after administration. Eyelids were removed and frozen on dry ice and then stored at -80°C until P-AKT ELISA analysis was performed.

[0156] exist Figure 20 Animals were administered 10 μl of eye drops containing 1.27 mg / ml IGF-1LR3 (SEQ ID NO: 12) or 1 mg / ml IGF-1E3R (SEQ ID NO: 4) at equimolar concentrations based on molecular weight and then sacrificed as previously described 1 hour after administration. Eyelids were removed and frozen on dry ice and then stored at -80°C until P-AKT ELISA analysis was performed.

[0157] exist Figure 21 Animals were administered 10 μl of eye drops containing 0.3, 1 or 3 mg / ml IGF-1LR3 (SEQ ID NO: 12) daily for 2 weeks and were sacrificed 24 hours after the last dose.

[0158] exist Figure 22A and 22B Animals received a 10 μl dose of vehicle (PBS) or 1 mg / ml IGF-1LR3 (SEQ ID NO: 12) bilaterally in each eye daily for 4 weeks.

[0159] exist Figure 23 Animals were administered 10 μl of eye drops containing 1.27 mg / ml IGF-1LR3 (SEQ ID NO: 12) or 1 mg / ml IGF-1E3R (SEQ ID NO: 4) at equimolar concentrations based on molecular weight daily for 2 weeks and were sacrificed 24 hours after the last dose.

[0160] Tissue collection, processing, and analysis

[0161] Mice were killed by cervical dislocation under isoflurane anesthesia, and the left and right upper eyelids were removed. The eyelids were then trimmed to the central 4 mm of tissue and embedded in tissue- The tissue blocks were then sliced ​​into 8 μm thick sections using a Leica CM 1850 cryostat (Leica, Wetzlar, Germany). The tissue sections were then stored in an ultra-low temperature freezer until processed for fluorescence microscopy.

[0162] To measure the basal proliferation rate or the effect of IGF-1LR3 on cell proliferation ( Figure 13A 、 Figure 13B 、 Figure 14A and Figure 14B ), the tissue sections were stained with the cell proliferation marker Ki67. Rabbit anti-Ki67 antibody (Abcam, catalog number #ab15580) was reacted with the tissue sections in a humidified box at 37 ° C for 1 hour. The sections were then washed with PBS (3 times, 5 min each time), and the secondary antibody (goat anti-rabbit AlexaFluor546, Invitrogen) was applied to the sections for 1 hour, followed by rinsing with PBS (3 times, 5 min each time), counterstained with DAPI (1:5000), and then covered with a coverslip. Fluorescence imaging was performed on a Leica DMI6000B fully automatic inverted fluorescence microscope (Leica Microsystems Inc., Buffalo Grove, IL), and tiled images of eyelid tissue sections and meibomian glands were collected using a low light camera (QIClick, QImaging, British Columbia, Canada) and a Leica 20×HC Plan Apo, 0.75NA objective lens. The images were then stitched using MetaImaging Series software. Ki67 labeling was quantified using QuPath, a bioimaging software analysis tool for high-throughput biomarker analysis. Initially, individual acini in each tissue section were annotated using the free-form region of interest tool, and the positive cell detection tool was used to identify Ki67-positive nuclei within each acinus. The number of Ki67-positive cells and the perimeter of each acinus were then recorded, and the average number of labeled cells / 100 μm of acinar perimeter was calculated for each section. The average of the three tissue sections was then determined, and the average across mice was calculated. The Student's t-test ( Figure 13A and Figure 13B ) or two-way ANOVA, Tukey, and all pairwise multiple comparison procedures in SigmaStat (Systat Software Inc., Point Richmond, CA) and GraphPad Prism (Insight Partners, NY, NY) were used to determine differences between treatment groups.

[0163] To assess changes in lipid synthesis ( Figure 16A and Figure 16B ), cells were stained with the neutral lipid fluorescent probe HCS LipidTox (Invitrogen, Carlsbad, CA). Cells were initially fixed with 2% paraformaldehyde in PBS, then rinsed and incubated in LipidTox solution (dilution 1:100) at room temperature for 20 minutes, followed by DAPI staining. Fluorescent staining was then imaged using a Leica DMI6000B inverted microscope, and tiled images on eyelid sections were collected and stitched together using MetaImaging software. To quantify newly synthesized lipid droplets near basal acinar cells, lipid-stained areas in the meibomian glands were segmented using a threshold subroutine to identify pixels with an intensity >50. Threshold regions were then extracted using the repeating plane function, and newly synthesized individual lipid droplets showing high-intensity fluorescent staining were identified using a set of counting subroutines to identify particles with a diameter greater than 2 μm and less than 5 μm (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. Then the number of lipid droplets / acinus was calculated to be 4000 μm 2 The mean acinar area was normalized. The mean number of lipid droplets was then calculated in three separate sections, followed by the mean number per mouse. Differences in the number of droplets per acinus within each treatment group were then determined using one-way ANOVA with Tukey's method and all pairwise multiple comparisons performed in GraphPad Prism (InsightPartners, NY, NY).

[0164] To evaluate the activation of IGF-1LR3 on IGF1R1 ( Figure 17), phosphorylated Akt levels were measured using the pan-specific DuoSetIC ELISA for phosphorylated Akt (S473) (RnD# DYC887B-2). Eyelid tissue was snap-frozen on dry ice and stored at -80°C until subsequent analysis. Samples were weighed and homogenized using a NextAdvance Bullet Blender Gold (BB24AU) and a green, dark blue, or red Eppendorf lysis kit (NextAdvance) with 1 ml of T-PER (Thermo Scientific, 78510) per 50 mg of tissue, containing protease and phosphatase inhibitors (Thermo Scientific, A32959). The homogenate was then used directly for the assay. The ELISA was performed in a 384-well plate with slight modifications according to the manufacturer's recommendations. Briefly, wells of a 384-well plate were coated with 25 μl of 6 μg / ml phospho-Akt1 (S473) capture antibody (#841692) in PBS (25 μl / 384 well), sealed, and left 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 h 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 h at room temperature. The 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 phospho-Akt1 (S473) detection antibody (#843081) diluted in PBS containing 1% BSA for 1 h 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 streptavidin-HRP A (RnD#890803) diluted 1:200 in PBS containing 1% BSA for 20 min 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 TMB substrate solution (Abcam TMB ELISA Substrate High Sensitivity (ab171523; Lot No. GR3427893-1)) for 10-20 min at room temperature. 12.5 μl of stop solution (RnD#895926 from Auxiliary Kit 2) was added. A Promega GloMAX Discover microplate reader was used to collect the standard curve and OD450 data for each sample.Sample data were interpolated from the standard curve, corrected for the dilution factor, and plotted, and pAKT levels were graphed in GraphPad Prism (InsightPartners, NY, NY) and statistically compared using one-way ANOVA and Tukey's method for multiple comparisons.

[0165] Imaging of the mouse eyelid meibomian glands

[0166] Before the start of the study and before sacrifice, transilluminated meibomian gland imaging was performed using a previously published technique ( Figure 15A and Figure 15B ). Briefly, mice were anesthetized with isoflurane and placed under a binocular dissecting microscope (Leica MZ16FA, Leica Microsystems, Heerbrugg, Switzerland) equipped with a monochrome camera (DFC340FX, Leica Microsystems, Heerbrugg, Switzerland). Meibomian gland imaging was performed by transilluminating the eyelids using a broadband halogen light source (OSL2 and OSL2B, Thorlabs, Newton, NJ). The light source was transmitted by a special optical fiber (BFL200HS02, Thorlabs, Newton, NJ) having an input connector consisting of a fiber bundle of seven 200 μm in diameter and an output connector consisting of a linear array of optical fibers connected to a diffuser and a prism by glue. All images were taken at 20 times magnification. After sacrifice, the eyelids were removed and ex vivo meibomian gland imaging was performed by placing the eyelids on an LED backlight plate and taking transillumination photographs with a dissecting microscope. To quantify meibomian gland area, individual eyelid images were analyzed using Meta Imaging Series software (Molecular Devices, Downington, PA). Specifically, the area of ​​each meibomian gland was manually outlined, and the area of ​​the region of interest for each gland in each image was measured. The mean area of ​​each eyelid gland was then calculated, and differences between groups were statistically analyzed using a one-tailed Mann-Whitney test using GraphPad Prism (Insight Partners, NY, NY).

[0167] Immunohistochemistry (IHC)

[0168] Eyelids from wild-type (WT) female C57BL / 6J mice (Jackson Labs, strain #000664) were dissected. Tissues were fixed overnight in 4% paraformaldehyde and replaced with 30% sucrose solution 24 hours later. Eyelids were then embedded in OCT medium, cut into 10-micron sections, and mounted on 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 Alexa Fluor-labeled secondary antibody, and for IGFBP2 using 1 μg / mL rabbit anti-IGFBP2 (Abcam #ab188200) followed by detection with an anti-rabbit Alexa Fluor-labeled secondary antibody. During the antibody incubation period, slides were washed three times with PBS + 0.1% Tween-20. Slides were then coverslipped and imaged.

[0169] exist Figure 21 and Figure 23 In the experiment, animals were sacrificed as previously described, and the eyelids were fixed in 4% paraformaldehyde overnight and replaced with 30% sucrose solution 24 hours later. The eyelids were then embedded in OCT culture medium, cut into 10 micron sections, and attached to slides. The tissue was then stained for Ki67 using 1 ug / mL rat anti-Ki67 antibody (Invitrogen #14-5698-82), followed by detection using an anti-rat Alexafluor labeled secondary antibody. During the antibody incubation period, the slides were washed 3 times with PBS + 0.1% Tween-20. The slides were then covered with a coverslip and imaged. The cell nuclei were then stained with Hoescht and covered with a coverslip for imaging. Random regions of interest (ROIs) containing meibomian gland alveoli were determined. The perimeter of the alveoli was outlined based on Hoescht staining in Image J, and the number of Ki67+ cells on the inner wall of the perimeter was measured and expressed as the number of Ki67+ cells per 100 μm of the perimeter of the alveoli. Data were then quantified and analyzed in GraphPad Prism (Insight Partners, NY, NY).

[0170] result

[0171] like Figure 13A and Figure 13B As depicted, meibomian gland acini in young mice showed increased proliferation compared to old mice. Systemic IGF-1LR3 treatment resulted in increased proliferation in meibomian gland acini in aged mice ( Figure 14A and Figure 14B ); atrophy was reversed and meibomian gland area increased in aged mice ( Figure 15A and Figure 15B ); regardless of age, lipid synthesis in the meibomian glands increases ( Figure 16A and Figure 16B ); and can be delivered systemically or via eye drops to activate IGF1R in the eyelid ( Figure 17 ).

[0172] Next, the duration of the effects of the IGF-1 variants was investigated. IGF-1LR3 (SEQ ID NO: 12) maintained significantly elevated pAKT (IGF1R activation) 2 hours after administration, whereas wild-type IGF-1 no longer showed significant pAKT 2 hours after administration, as shown in Table 1. Figure 18 Compared with wild-type IGF-1, IGF-1LR3 (SEQ ID NO: 12) and another IGFBP binding-deficient IGF-1 variant, IGF-1Des1-3 (SEQ ID NO: 3), significantly increased pAKT (IGF1R activation) 2 hours after administration, as shown in Figure 2. Figure 19 This suggests that some IGF-1 variants with reduced affinity for IGFBPs prolong the duration of IGF1R activation compared to wild-type IGF-1. Furthermore, IGF-1LR3 (SEQ ID NO: 12) and IGF-1E3R (SEQ ID NO: 4) have similar potency in vivo. IGF-1LR3 (SEQ ID NO: 12) or the IGFBP-binding-deficient IGF-1 variant IGF-1E3R (SEQ ID NO: 4) increased pAKT (IGF1R activation) to levels comparable to IGF-1 at 1 hour after administration, as shown in Figure 5. Figure 20 shown.

[0173] Next, we tested the effects of IGF-1LR3 and IGF-1ER3 on the meibomian glands of aged mice. IGF-1LR3 (SEQ ID NO: 12) induced a dose-responsive proliferation of basal cells in the meibomian glands, as Figure 21 Daily administration of IGF-1LR3 (SEQ ID NO: 12) at the indicated concentrations of 0.3, 1, or 3 mg / ml for two weeks induced basal cell proliferation as determined by IHC. Figure 21 As shown. IGF-1LR3 (SEQ ID NO: 12) regenerates the atrophic meibomian glands of aging mice. After daily treatment with IGF-1LR3 (SEQ ID NO: 12) for one month, the area of ​​the meibomian glands increased compared to before and after treatment, as shown in Figure 2. Figure 22A and Figure 22BAs shown. IGF-1LR3 (SEQ ID NO: 12) and IGF-1E3R (SEQ ID NO: 4) induce basal cell proliferation in the meibomian gland. Daily eye drop administration of IGF-1LR3 (SEQ ID NO: 12) or IGF-1E3R (SEQ ID NO: 4) for two weeks induced basal cell proliferation in aged mice as determined by IHC, as shown Figure 23 shown.

[0174] Although preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that these embodiments are provided by way of example only. Many variations, modifications, and substitutions will now occur to those skilled in the art without departing from the present invention. It should be understood that various alternatives to the embodiments of the present invention described herein may be employed in practicing the present invention. The following claims are intended to define the scope of the invention, and methods and structures within the scope of these claims and their equivalents are intended to be encompassed therein.

Claims

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

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

3. A pharmaceutical composition comprising a therapeutically effective amount of an IGF-1 variant, wherein topical application of the pharmaceutical composition to the eye or eyelid results in one or more of the following: (a) Increased size of the meibomian glands; (b) Meibomian gland atrophy and reduction; (c) reversal of age-related meibomian gland atrophy; (d) increased function of one or more meibomian gland cells; (e) increased corneal epithelial cell proliferation; (f) Increased corneal healing rate; (g) increased activation of the IGF1 receptor (IGF1R) in the meibomian glands; (h) increasing the duration of IGF1R activation in the meibomian glands; and (i) The lipid content of the meibomian glands is increased.

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

5. The pharmaceutical composition of any one of the preceding claims, 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 according to any one of the preceding claims, wherein the pharmaceutical composition is formulated for delivery via eye drops.

7. The pharmaceutical composition of any one of claims 1-5, wherein the pharmaceutical composition comprises a cream for application to one or both eyelids of a subject.

8. The pharmaceutical composition of any one of the preceding claims, wherein when the pharmaceutical composition is administered to a subject suffering from meibomian gland dysfunction, the pharmaceutical composition 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.

9. The pharmaceutical composition of any one of the preceding claims, wherein when the pharmaceutical composition is administered to a subject suffering from meibomian gland dysfunction, the pharmaceutical composition results in an increase in the median lipid content within the subject's meibomian glands compared to a subject not receiving the pharmaceutical composition.

10. The pharmaceutical composition of any one of the preceding claims, wherein when the pharmaceutical composition is administered to a subject suffering from meibomian gland dysfunction, the pharmaceutical composition results in an increase in the median lipid mass within the subject's meibomian glands compared to a subject not receiving the pharmaceutical composition.

11. The pharmaceutical composition of any one of the preceding claims, wherein when the pharmaceutical composition is administered to a subject suffering from meibomian gland dysfunction, the pharmaceutical composition results in an increase in median lipid release from alveoli of the subject's meibomian glands compared to a subject not receiving the pharmaceutical composition.

12. The pharmaceutical composition of any one of the preceding claims, wherein when the pharmaceutical composition is administered to a subject with meibomian gland dysfunction, the pharmaceutical composition results in an increase in the median duration of Akt phosphorylation in meibomian gland cells relative to a subject not receiving the pharmaceutical composition.

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

14. The pharmaceutical composition according to any one of claims 1 to 12, wherein the 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 1-13, wherein the IGF-1 variant comprises one or more amino acid substitutions relative to wild-type IGF-1 (SEQ ID NO: 1).

16. The pharmaceutical composition of any one of claims 1-13 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 position numbering is based on an alignment of the IGF-1 variant with SEQ ID NO: 1, wherein positions are numbered from the N-terminus of SEQ ID NO: 1 to the C-terminus of SEQ ID NO: 1, starting from position 1 of the N-terminus.

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

6.

18. The pharmaceutical composition of any one of claims 1-12 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 position numbering is based on an alignment of the IGF-1 variant with SEQ ID NO: 1, wherein positions are numbered from the N-terminus of SEQ ID NO: 1 to the C-terminus of SEQ ID NO: 1, starting from position 1 of the N-terminus.

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

2.

20. The pharmaceutical composition of any one of claims 1-12 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 position numbering is based on an alignment of the IGF-1 variant with SEQ ID NO: 1, wherein positions are numbered from the N-terminus of SEQ ID NO: 1 to the C-terminus of SEQ ID NO: 1, starting from position 1 of the N-terminus.

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

4.

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

5.

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

7.

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

12.

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

26. The pharmaceutical composition of claim 25, wherein the IGF-1 variant is coupled to a cell penetrating peptide selected from the group consisting of SEQ ID NO: 10 and SEQ ID NO:

11.

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

9.

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

8.

29. A pharmaceutical composition according to any preceding claim, further comprising one or more pharmaceutically acceptable excipients.

30. The pharmaceutical composition of claim 29, wherein the one or more pharmaceutically acceptable excipients comprises one or more of water, saline, sucrose, lactose, malic acid, cellulosic sugars, mannitol, maltitol, dextran, sorbitol, starch, agar, alginate, chitin, chitosan, pectin, tragacanth, gum arabic, gelatin, collagen, casein, albumin, a synthetic or semisynthetic polymer or glyceride, methylcellulose, hydroxypropyl methylcellulose, and polyvinylpyrrolidone.

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

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

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

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

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

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

37. The pharmaceutical composition of any preceding claim, wherein administration of the pharmaceutical composition to the eye or eyelid of the subject results in an increase in the size of the meibomian glands.

38. The pharmaceutical composition of any preceding claim, wherein administration of the pharmaceutical composition to the eye or eyelid of the subject results in reduced meibomian gland atrophy.

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

40. The pharmaceutical composition of any preceding claim, wherein administration of the pharmaceutical composition to the eye or eyelid of the subject results in increased function of one or more meibomian gland cells.

41. The pharmaceutical composition of any preceding claim, wherein administration of the pharmaceutical composition to the eye or eyelid of the subject results in increased corneal epithelial cell proliferation.

42. The pharmaceutical composition of any preceding claim, wherein administration of the pharmaceutical composition to the eye or eyelid of the subject results in increased corneal healing.

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

44. The pharmaceutical composition of any preceding claim, wherein administration of the pharmaceutical composition to the eye or eyelid of the subject results in an increase in the duration of IGF1R activation in the meibomian gland.

45. The pharmaceutical composition of any preceding claim, 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 glands.

46. ​​The pharmaceutical composition according to any preceding claim, wherein when the pharmaceutical composition is administered to spheroids of IHGMGE cells, the pharmaceutical composition results in an increase in lipid content in the spheroids on average.

47. A kit comprising: (a) the pharmaceutical composition of any one of claims 1-6 and 1-46; and (b) an eye dropper for delivering the pharmaceutical composition as eye drops.

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

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

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

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

52. The method of claim 51, wherein the pharmaceutical composition is administered to the subject's eyes via eye drops.

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

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

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

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

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

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

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

60. The method of any one of claims 48-59, wherein the method does not comprise the administration of any other phospholipid deposition inducing agent.

61. The method of any one of claims 48-60, wherein the method does not comprise administering one or both of azithromycin and doxycycline.

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

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

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

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

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

67. The pharmaceutical composition of claim 65, wherein the pharmaceutical composition comprises a cream for application to one or both eyelids.

68. The pharmaceutical composition of claim 65, wherein the pharmaceutical composition is formulated for systemic delivery.

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

70. The pharmaceutical composition of any one of claims 65-69, 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. The pharmaceutical composition of any one of claims 65-70, wherein administration of the pharmaceutical composition to a subject suffering from meibomian gland dysfunction results in an increase in the median lipid content within the meibomian glands.

72. The pharmaceutical composition of any one of claims 65-71, wherein administration of 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.

73. The pharmaceutical composition of any one of claims 65-72, wherein administration of 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.

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

75. The pharmaceutical composition according to any one of claims 65-74, wherein the pharmaceutical composition does not comprise any other phospholipid deposition inducing agent.

76. The pharmaceutical composition of any one of claims 65-75, wherein the pharmaceutical composition does not comprise either azithromycin or doxycycline.

77. The pharmaceutical composition of any one of claims 65-76, wherein the one or more pharmaceutically acceptable excipients comprises one or more of water, saline, sucrose, lactose, malic acid, cellulosic sugars, mannitol, maltitol, dextran, sorbitol, starch, agar, alginate, chitin, chitosan, pectin, tragacanth, gum arabic, gelatin, collagen, casein, albumin, a synthetic or semisynthetic polymer or glyceride, methylcellulose, hydroxypropyl methylcellulose, and polyvinylpyrrolidone.

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

1.

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

2.

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

3.

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

4.

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

5.

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

6.

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

7.

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

8.

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

12.

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

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

9.

90. A kit comprising: (a) the pharmaceutical composition of any one of claims 65-66 and 68-89; and (b) an eye dropper for delivering the pharmaceutical composition as eye drops.

91. A method for treating an eye 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.

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

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

94. The method of claim 93, wherein the pharmaceutical composition is administered to the subject's eye via an eye dropper.

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

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

97. The method of any one of claims 91-96, wherein administering the pharmaceutical composition to the subject results in a median increase in the surface area or volume of the meibomian glands of the inner eyelid surface.

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

99. The method of any one of claims 91-98, wherein administering the pharmaceutical composition to the subject suffering from meibomian gland dysfunction results in increased lipid release from the alveoli of the meibomian glands.

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

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

102. The method of any one of claims 91-101, wherein the method does not comprise the administration of any other phospholipid deposition inducing agent.

103. The method of any one of claims 91-102, wherein the method does not comprise administering either azithromycin or doxycycline.

104. The method of any one of claims 91-103, wherein the eye condition comprises dry eye.

105. The method of any one of claims 91-103, wherein the eye condition comprises meibomian gland dysfunction.

106. The method of any one of claims 91-103, wherein the eye disorder comprises Sjögren's syndrome.

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