Ophthalmic polypeptide compositions for the treatment of posterior eye disorders

Abstract

The present disclosure is in the field of ophthalmic compositions comprising synthetic polypeptides suspended, or dispersed, in a semifluorinated alkane vehicle, which are useful in the treatment ophthalmic disorders, particularly disorders of the posterior eye and retina, including diabetic retinopathy. In particular, the disclosure pertains to synthetic peptides having activity as nitric oxide synthase inhibitors.

Description

OPHTHALMIC POLYPEPTIDE COMPOSITIONS FOR THE TREATMENT OF POSTERIOR EYE DISORDERSFIELD

[0001] The present disclosure is in the field of ophthalmic compositions comprising synthetic polypeptides suspended, or dispersed, in a semifluorinated alkane vehicle, which are useful in the treatment of ophthalmic disorders, particularly disorders of the posterior eye and retina, including diabetic retinopathy. In particular, the disclosure pertains to synthetic peptides having activity as nitric oxide synthase inhibitors.BACKGROUND

[0002] The posterior segment or posterior cavity of the eye is the back two-thirds of the eye, including the anterior hyaloid membrane and all of the optical structures behind it: the vitreous humor, retina, choroid, and optic nerve. The posterior segment should not be confused with the posterior chamber, which is the small space directly posterior to the iris but anterior to the lens, and which is part of the anterior segment of the eye. The eye also has three chambers: anterior, posterior, and vitreous. Both the anterior and posterior chambers are located within the anterior segment, while the vitreous chamber is located in the posterior segment. The vitreous chamber is the largest of the three chambers and is located behind the lens and in front of the optic nerve. This chamber is filled with a thick, clear gel-like substance called the vitreous humor (also vitreous body). The vitreous humor plays a crucial role in supporting the posterior side of the lens.

[0003] Posterior segment eye diseases (PSED) are becoming increasingly common. The three leading PSED are glaucoma, age-related macular degeneration (ARMD), and diabetic retinopathy (DR). These three diseases are now recognized as a major cause of visual impairment worldwide and are more prevalent than infectious causes of visual impairment, such as trachoma and corneal ulcers. Other PSED include retinal vein occlusion, ischemic optic neuropathy (ION), ocular hypertension (a precursor to glaucoma), macular edema, uveitis, and endophthalmitis.

[0004] Diabetic retinopathy is an eye condition that can cause vision loss and blindness in people who have diabetes (diabetes mellitus). It affects blood vessels in the retina. The retina is a thinlayer of light-sensitive tissue on the back wall of the eye. The optical system of the eye focuses light on the retina much like light is focused on the film in a camera. The retina translates that focused image into neural impulses and sends them to the brain via the optic nerve.

[0005] Diabetic retinopathy is caused by prolonged high blood glucose damaging the small blood vessels of the retina, although the precise mechanism by which this occurs is unknown. Progression of diabetic retinopathy is accompanied by loss of capillary cells, increased blood vessel permeability in the retina, and altered retinal blood flow, all of which can reduce the amount of blood oxygen that gets delivered to the retina. Poor oxygenation of tissues drives the formation of new blood vessels throughout the retina, resulting in the proliferative stage of disease. These new blood vessels tend to rupture easily, causing bleeding within the eye, scarring, and damage to the retina or macula. Recent evidence suggests an association between diabetic retinopathy and inflammation.

[0006] The earliest changes leading to diabetic retinopathy include narrowing of the retinal arteries associated with reduced retinal blood flow; dysfunction of the neurons of the inner retina, followed in later stages by changes in the function of the outer retina, associated with subtle changes in visual function; dysfunction of the blood-retinal barrier, which protects the retina from many substances in the blood (including toxins and immune cells), leading to the leaking of blood constituents into the retinal neuropile. Later, the basement membrane of the retinal blood vessels thickens, capillaries degenerate and lose cells, particularly pericytes and vascular smooth muscle cells. This leads to loss of blood flow and progressive ischemia, and microaneurysms which appear as balloon-like structures jutting out from the capillary walls, which recruit inflammatory cells; and advanced dysfunction and degeneration of the neurons and glial cells of the retina. The condition typically develops about 10-15 years after receiving the diagnosis of diabetes mellitus.

[0007] Diabetic retinopathy affects up to 80 percent of those who have had both type 1 and type 2 diabetes for 20 years or more.

[0008] Nearly all people with diabetes develop some degree of retinal damage over several decades with the disease. In the early stages of disease, retinal damage can only be detected by a retinal exam, and has no noticeable effect on vision. Early signs include small bulges in retinal blood vessels called microaneurysms. Then larger abnormalities in retinal vessels: cotton wool spots, hemorrhages, lipid deposits called “hard exudates,” intraretinal microvascularabnormalities, and abnormal-looking retinal veins. Eventually, patients progress to a stage where new blood vessels grow throughout the retina (proliferative diabetic retinopathy), and these vessels often bleed, causing dark floating spots obstructing vision. Major bleeding can completely block vision.

[0009] Around half of people with diabetic retinopathy develop swelling of the macula, which is called macular edema, and this can begin at any time. Macular edema occurs when fluid and protein deposits collect on or under the macula of the eye (a yellow central area of the retina) and causes it to thicken and swell. The swelling may distort a person’s central vision, because the macula holds tightly packed cone cells, which provide sharp, clear, central vision to enable a person to see detail, form, and color that is directly in the center of the field of view. If the swelling occurs near the center of the macula, it can cause vision disruptions ranging from mildly blurred vision to severe loss of the center of an affected person's visual field. Left untreated, around 30% of those with macular edema experience vision disruption over the next 3-5 years. Diabetic macular edema (DME) is the most common cause of visual loss in both proliferative, and non-proliferative diabetic retinopathy (NPDR).

[0010] The repeated processes of blood vessel growth, swelling, and scarring can eventually cause retinal detachment, which manifests as the sudden appearance of dark floating spots, flashes of light, or blurred vision. This is a form of tractional retinal detachment, which is a retinal detachment caused by fibrovascular tissue (scar tissue) pulling the sensory retina from the retinal pigment epithelium.

[0011] Neovascular glaucoma, an uncommon type of glaucoma, is often caused by proliferative diabetic retinopathy (PDR), and it is difficult or nearly impossible to treat. Individuals with poor blood flow to the eye are highly at risk for this condition. Neovascular glaucoma results when new, abnormal vessels begin developing in the angle of the eye that begin blocking drainage. Patients may begin to rapidly lose their eyesight.

[0012] The American Academy of Ophthalmology (AAO) divides diabetic retinopathy into five categories of progressive severity. The first category, “no apparent retinopathy,” describes those with a healthy retinal exam. The next three categories, mild, moderate, and severe nonproliferative diabetic retinopathy (NPDR), describe increasing levels of damage to the retina. People with mild NPDR have microaneurysms in the retina, but no other damage. Those with severe NPDR have 20 or more retinal hemorrhages in each quadrant of the retina, a distinctivepatern of damage on the veins of the retina called “venous beading” in at least two retinal quadrants, and obvious intraretinal microvascular abnormalities anywhere on the retina. Moderate NPDR is defined as more severe than mild NPDR, but not yet meeting the criteria for severe NPDR. The fifth stage, proliferative diabetic retinopathy (PDR), is for those with new blood vessels forming throughout the retina (retinal neovascularization), or blood leaking into the vitreous humor (vitreous hemorrhage), or between the vitreous membrane and retina (preretinal hemorrhage).

[0013] Similar guidelines separately divide macular edema into two categories: “macular edema apparently absent” and “macular edema apparently present.” The latter is further subdivided into “mild” — retinal thickening or lipid deposits far from the macula's center; “moderate” — thickening or deposits near the center; and “severe” — thickening or deposits on the macula center. Optical coherence tomography is frequently used to assess macular edema.

[0014] Fluorescein angiography is often used by retina specialists to further assess diabetic retinopathy severity and to determine sites of macular damage.

[0015] Diabetic retinopathy can occur in all types of diabetic patients, including those with type 1 diabetes, type 2 diabetes, and gestational diabetes. Around 35% of people with diabetes have some kind of diabetic retinopathy, and around 10% experience some degree of vision loss. Diabetic retinopathy is particularly common in those with type 1 diabetes - affecting 25% of people five years from diagnosis, 60% 10 years from diagnosis, and 80% 15 years from diagnosis. The chances of disease progression are heavily influenced by blood sugar control, but on average 7% of those with diabetes experiencing proliferative diabetic retinopathy and 7% diabetic macular edema. Diabetic retinopathy is the leading cause of vision loss in those 20-74 years old.

[0016] The global burden of diabetic retinopathy increased dramatically from 1990 to 2015 — from 1.4 million to 2.6 million people with visual impairment; from 0.2 million to 0.4 million blinded — due in large part to the increasing burden of type 2 diabetes in low- and middle-income countries.

[0017] The major risk factors for developing diabetic retinopathy are duration of diabetes, effectiveness of blood glucose control, and, to a lesser extent, hypertension. Five years after diabetes diagnosis, around 25% of those with type 1 diabetes have some degree of diabetic retinopathy, while 2% have proliferative diabetic retinopathy. By 15 years after diagnosis, thatincreases to 80% with some retinopathy, and 25% with proliferative disease. Children are an exception — regardless of duration of diabetes, children rarely experience sight-threatening retinopathy; however, puberty can accelerate retinopathy progression. Pregnancy can also accelerate the progression of diabetic retinopathy (although women with gestational diabetes are not at risk).

[0018] Both chronically high blood sugar (measured by a high HbAlc) and highly variable blood sugar are associated with developing diabetic retinopathy. Several more minor risk factors also exacerbate diabetic retinopathy, namely kidney disease, abnormal blood lipids, high body mass index, and smoking.

[0019] There is also evidence of genetic predisposition to diabetic retinopathy in type 2 diabetes, connected to variations in genes associated with diabetic retinopathy, glucose, low-density lipoprotein cholesterol, and systolic blood pressure. In particular, several variations in the VEGFC gene (encoding vascular endothelial growth factor C, VEGF-C) have also been associated with an increased risk of developing macular edema.

[0020] VEGF-C is a protein that is a member of the platelet-derived growth factor / vascular endothelial growth factor (PDGF / VEGF) family. The main function of VEGF-C is to promote the growth of lymphatic vessels (lymphangiogenesis), acting on lymphatic endothelial cells (LECs) primarily via its receptor VEGFR-3. However, in addition to its effect on lymphatic vessels, it can also promote the growth of blood vessels and regulate their permeability, mediated either by primary receptor VEGFR-3 or its secondary receptor VEGFR-2.

[0021] There are four common treatments for diabetic retinopathy: anti-VEGF injections (such as aflibercept, bevacizumab and ranibizumab), steroid injections (such as triamcinolone), pan- retinal laser photocoagulation, and vitrectomy. Such injections are given intravitreally.

[0022] Treating people with DME with intravitreal injections of triamcinolone may lead to some degree of improvement in visual acuity as the steroid decreases the macular edema (thickening of the retina at the macula) but the effect of triamcinolone is not permanent. It may only last up to three months, which necessitates repeated injections for maintaining the beneficial effect, and complications may include cataract, steroid-induced glaucoma, and endophthalmitis.

[0023] High intravitreal drug concentrations are required in the treatment of posterior segment eye diseases. However, the anatomy, physiology, and biochemistry of the eye make the eye resistant to significant concentrations of foreign substances. An understanding of thecharacteristics of the blood-eye barrier is important in efforts to achieve drug delivery for ophthalmic diseases. This barrier, which compartmentalizes the eye, is maintained by tight junctions at the retinal vascular endothelium, the iris vascular epithelium, and the nonpigmented ciliary epithelium. It comprises two components: an outer component formed by junctional complexes of the retinal pigmented epithelium (RPE) and the pigment epithelial cells of the pars plana, and an inner component formed by tight junctions between endothelial cells in the retinal capillaries. The barrier blocks pathogens from reaching ocular tissues but also hinders systemic pharmacologic agents from reaching potential targets inside the eye. It also reduces convection of molecules because it has no cellular components and is selectively permeable to more lipophilic molecules. Because of this, many strategies developed to deliver treatment for posterior segment disease have failed to show clinical efficacy.

[0024] Topical drug application, the most common method of ocular drug delivery, is useful in the treatment of many anterior segment disorders. This noninvasive mode of drug delivery selectively targets the anterior chamber structures; however, the cornea represents a significant barrier for efficient drug delivery. The corneal epithelium is a lipophilic tissue and contributes to a major reduction in penetration by hydrophilic drugs; less than 5% of the total administered topical dose reaches the aqueous humor, and far less penetrates into the posterior segment. A major fraction of drug following topical administration is lost through lacrimation, tear dilution, nasolacrimal drainage, and tear turnover. Such precorneal losses result in very low ocular bioavailability.

[0025] A synthetic polypeptide of 19 amino acids, called PnTx(19), has been disclosed, which is constituted from the sequence of the native toxin PnTx2-6 of the Phoneutria nigriventer spider. See U.S. 9,279,004. This spider, popularly known as “aranha-armadeira” (“armed spider”), produces a venom which is rich in bioactive polypeptides with different pharmacological effects, acting chiefly on ion channels and receptors. Studies have demonstrated that the venom toxicity comes largely from the effects of a peptide fraction which include PnTx2-6, and purified PnTx2- 6 reproduces the symptomatic effects of the total fraction after intracerebral injection into mice. PnTx2-6 has been shown to have several mechanisms of action, including effects on sodium channels, glutamate release, and nitric oxide synthase activity.

[0026] U.S. 9,279,004 discloses the synthetic peptide PnTx(19) (also known as PnPP-19), having the sequence GERRQYFWIAWYKLANSKK (SEQ ID NO. 1) (N-Gly-Glu-Arg-Arg-Gln-Tyr-Phe-Trp-Ile-Ala-Trp-Tyr-Lys-Leu-Ala-Asn-Ser-Lys-Lys-C), which was synthesized chemically by using the Fmoc / t-butyl strategy of synthesis on solid support. The peptide has 19 amino acid residues, is linear and was designed from the probable three-dimensional structure of the PnTx2-6 by mimicking the hydrophobic “core” and the positively charted residues that surround this region. The sequence (discontinuous epitope) developed was modified from the natural sequence by replacing cysteine- 17 with serine, and amidating the C-terminal portion and acetylating the N-terminal portion. U.S. 9,279,004 discloses pharmaceutical compositions containing this peptide for the treatment of erectile dysfunction and / or in potentiating the erectile function.

[0027] C-terminal amidation and N-terminal acetylation are common strategies for working with bioactive peptides, because these modifications increase the stability of the peptides, especially towards enzymatic degradation by aminopeptidases, exopeptidases, and synthetases, while retaining the fundamental sequence of the peptide.

[0028] Subsequently, U.S. Patent Publication 2021 / 0060125 disclosed the use of PnTx(19) in the treatment of eye diseases, in particular, glaucoma and increased intraocular pressure. Intraocular pressure (IOP) is controlled by a delicate balance in the production and elimination of the aqueous humor (AH) in the anterior eye segment. The AH is produced and secreted by the ciliary epithelium in the posterior eye chamber. Under normal physiological conditions, the AH passes from the posterior to the anterior chamber through the pupil and is drained from the eye through the conventional (or trabecular) outflow, or through the unconventional (or uveoscleral) outflow pathways. Under normal physiological conditions, the conventional outflow mediates about 60%-90% of the AH drainage in humans, while the unconventional pathway plays a smaller role, which tends to become even smaller in older eyes. Increased resistance to outflow in the conventional pathway flow structures is the root cause of increased IOP. Recent evidence indicates that the nitric oxide (NO) signaling pathway has a role in ocular homeostasis, particularly regulating AH drainage and, therefore, IOP. In healthy human eyes, the capacity to form NO is found in the anterior ocular tissues. Nitric oxide (NO) has thus gained attention as a potential new target for the treatment of glaucoma because the biologic effects of NO could mediate increased AH drainage through the conventional outflow pathway. In addition, NO may also be important in maintaining basal blood flow to the retina and optic nerve. US 2021 / 0060125 therefore discloses the use of synthetic peptides including PnTx(19) for reducingintraocular pressure, treating or preventing eye diseases including ocular hypertension, ischemic optic neuropathy, glaucoma, age-related macular degeneration, and diabetic neuropathy.

[0029] US 2021 / 0060125 also discloses that while therapeutic peptides have shown great promise as novel therapeutics in the treatment of ocular diseases, their susceptibility to physical and chemical degradation, short in vivo half-lives, clearance by the mononuclear phagocytes (MPS) of the reticulum endothelial system (RES), risk of immunogenicity, and failure to permeate cell membranes pose high challenges to topic ocular administration of peptides. These barriers have warranted large efforts to allow the effective use of therapeutic peptides in the treatment of eye diseases. Indeed, there are only four protein drugs (three monoclonal antibodies, and one therapeutic protein) approved for the treatment of eye diseases, and they drugs are administered by intraocular injection. The repeated eye injections correlate with an increased propensity for complications, such as endophthalmitis, cataracts, retinal tears, and retinal detachment, thus representing a significant inconvenience for patients.

[0030] US 2021 / 0060125 discloses a variety of general strategies for formulation aqueous ophthalmic compositions for delivering the PnTx(19) / PnPP-19 peptide to the eye, preferably topical aqueous eye drops comprising ionic buffers, ionic tonicity modifying agents, polymeric viscosity modifying agents (e.g., polyacrylates, polyvinyl alcohols, modified celluloses, and other polar polymers), surfactants, and / or preservatives. Several animal studies are disclosed utilizing the peptide as a solution in saline (0.9%), and a phase I human clinical trial is described in which healthy patients are treated with peptide dissolved in 0.5% saline.

[0031] Such saline solutions of peptide would not be suitable for large-scale commercial manufacture and distribution as topical eye drops, however, because these vehicles would not provide sufficient long-term stability or protection from microbial contamination.

[0032] One of the major challenges associated with peptide formulation and delivery is maintaining the stability and form of the biological therapeutic, in particular for long-term storage and for transport. Much more so than small-molecule therapies, protein-based therapeutics are susceptible to physical and chemical instability under stress conditions such as temperature changes from freeze-thawing or during transport, exposure to light, oxygen or chemical / solvents, shear stress, and pH stress. They may undergo denaturation (e.g., loss of tertiary and / or secondary structure) or interact to form aggregates, especially in liquid formulations, and especially at high concentrations of peptide. High concentrations, however, arefrequently necessary to minimize dose volumes and to make administration more patientfriendly, especially for topical ophthalmic therapy.

[0033] Protein instability is particularly prevalent in aqueous solutions, and as a result, such products have limited shelf-life and / or requiring the development of complex cold-chain solutions. The alternative approach is the provision of the protein drugs as a lyophilized (i.e., freeze-dried) solid power form, but lyophilizates require careful and accurate reconstitution under sterile conditions in an aqueous media before use and thus are generally less convenient for patient and health-care provider use. The reconstitution step itself may trigger aggregation if the pH or temperature of the aqueous medium is suboptimal, the time allowed for rehydration is too short or the vial is too aggressively shaken during the dissolving step. The propensity for waste is also higher, as failure to properly dissolve the lyophilized antibody product within the recommended time period usually requires for the sample to be discarded.

[0034] Additional stabilizing excipients such as saccharides or polyols are often added to the pre-lyophilization peptide composition, along with other excipients such as bulking agents. The addition of other excipients may also be required after lyophilization in order to support the longer shelf-life of the peptide, adding to the number of components in the final formulation.

[0035] Ready-to-use liquid formulations would generally be preferred by the users, due to the ease of preparation for administration. If stable, a liquid formulation is also attractive for the pharmaceutical manufacturer due to the avoidance of lyophilization, which is time-consuming and costly both during drug development and routine manufacture. While many marketed biologic formulations are aqueous-based solutions, the pH of the medium can have a significant impact on the stability of the protein, and consequently, an optimized buffering system is always required, along with other formulation excipients such as antioxidant free-radical scavengers, surfactants and other anti-aggregation additives, or preservatives in order to provide stabilization to the antibody and counteract the various possible degradation processes that may occur during storage in an aqueous environment over time.

[0036] Alternative formulation options to lyophilization and aqueous solutions are also known, such as the use of non-aqueous liquids as carrier vehicles, particularly as suspensions in semifluorinated alkanes. WO2013 / 110621 (U.S. 9,757,460) describes the formulation of proteins and polypeptides in semifluorinated alkanes vehicles, including alpha-chymotrypsinogen A, alpha-chymotrypsin, bovine insulin, human insulin, bovine serum albumin, salmon calcitonin,and human calcitonin. W02015 / 011199 (U.S. 10,273,298) discloses antibodies and fusion proteins suspended in semifluorinated alkane vehicles, including bevacizumab, infliximab, etanercept, and certolizumab. WO2021 / 105392 (US 2022 / 0354786) discloses methods for preparing suspensions of protein particles in semifluorinated alkane vehicles, including lysozyme, bevacizumab, and a monoclonal antibody.

[0037] It is therefore an object of the present invention, to provide stable, non-aqueous, topical ophthalmic compositions comprising certain synthetic peptides in a semifluorinated alkane vehicle.BRIEF SUMMARY

[0038] In a first aspect, the present disclosure provides a pharmaceutical composition comprising a polypeptide, or a pharmaceutically acceptable salt thereof, dispersed, or suspended in a nonaqueous liquid vehicle comprising one or more semifluorinated alkanes, wherein the polypeptide is a polypeptide according to SEQ ID NO. 1, or an analog and / or derivative thereof.

[0039] In a second aspect, the present disclosure provides a pharmaceutical composition comprising a polypeptide, or a pharmaceutically acceptable salt thereof, dispersed, or suspended in a non-aqueous liquid vehicle comprising one or more semifluorinated alkanes, wherein the polypeptide is a polypeptide according to SEQ ID NO. 1, or an analog and / or derivative thereof, for use as a medicament or for use in a method of treating or preventing a posterior eye disorder (e.g., diabetic retinopathy).

[0040] In a third aspect, the present disclosure provides a method of treating or preventing a posterior eye disorder (e.g., diabetic retinopathy), comprising administering to a patient in need thereof a pharmaceutical composition comprising a polypeptide, or a pharmaceutically acceptable salt thereof, dispersed, or suspended in a non-aqueous liquid vehicle comprising one or more semifluorinated alkanes, wherein the polypeptide is a polypeptide according to SEQ ID NO. 1, or an analog and / or derivative thereof.

[0041] In a fourth aspect, the present disclosure provides a method of inducing nitric oxide synthase or increasing nitric oxide production in the tissues of the posterior eye (e.g., the retina, macula, and / or optic nerve), comprising administering to a patient in need thereof a pharmaceutical composition comprising a polypeptide, or a pharmaceutically acceptable salt thereof, dispersed, or suspended in a non-aqueous liquid vehicle comprising one or moresemifluorinated alkanes, wherein the polypeptide is a polypeptide according to SEQ ID NO. 1, or an analog and / or derivative thereof.

[0042] In a fifth aspect, the present disclosure provides a kit comprising a pharmaceutical composition comprising a polypeptide, or a pharmaceutically acceptable salt thereof, dispersed, or suspended in a non-aqueous liquid vehicle comprising one or more semifluorinated alkanes, wherein the polypeptide is a polypeptide according to SEQ ID NO. 1, or an analog and / or derivative thereof, and a container for holding the pharmaceutical composition and a drop dispenser for administering the composition, and optionally instructions for use of said composition as a medicament or for use in a method of treating or preventing a posterior eye disorder (e.g., diabetic retinopathy) or for use in a method of inducing nitric oxide synthase or increasing nitric oxide production in the tissues of the posterior eye.

[0043] In sixth aspect, the present disclosure provides a method of preparing a pharmaceutical composition comprising a polypeptide, or a pharmaceutically acceptable salt thereof, dispersed, or suspended in a non-aqueous liquid vehicle comprising one or more semifluorinated alkanes, wherein the polypeptide is a polypeptide according to SEQ ID NO. 1, or an analog and / or derivative thereof.

[0044] Further objects of the invention will become clear on the basis of the following description, examples, and patent claims.DETAILED DESCRIPTION

[0045] It has been unexpectedly discovered that some polypeptides disclosed herein, such as the polypeptide according to SEQ ID. NO. 1, may undergo enzymatic and / or non-enzymatic hydrolysis to form truncated analogs or metabolites, such as under physiological conditions. Without being bound by theory, it is believed that one or more of such truncated polypeptides may be active in inducing or promoting nitric oxide synthase activity. From the standpoint of regulatory approval, it is necessary to minimize unexpected hydrolysis and maximize chemical stability of a polypeptide formulated for pharmacologic use. Yet, the N-terminal and C-terminal modifications of such peptides disclosed in the art have proven inadequate to maximizing such stability, thus requiring improved formulations, such as non-aqueous formulations. At the same time, it may be beneficial to utilize shorter truncated sequences which are analogs and / orderivatives of SEQ ID NO.l in order to reduce unexpected enzymatic and / or non-enzymatic hydrolysis.

[0046] In a first aspect, the present disclosure provides a pharmaceutical composition (Composition 1) comprising a polypeptide, or a pharmaceutically acceptable salt thereof, dispersed, or suspended in a non-aqueous liquid vehicle comprising one or more semifluorinated alkanes, wherein the polypeptide is a polypeptide comprising SEQ ID NO. 1, or an analog and / or derivative thereof. In further embodiments of Composition 1, the present disclosure provides:1.1 Composition 1, wherein the polypeptide is the polypeptide according to SEQ ID NO. 1, without modification (derivatization of any amino acids);1.2 Composition 1, wherein the polypeptide is a derivative of the polypeptide according to SEQ ID NO. 1, wherein the polypeptide comprises one or more modifications of the N-terminal amino group, C-terminal carboxyl group, and / or modifications of the internal amino acid amino and / or carboxyl groups and / or hydroxyl groups (e.g., of the glutamic acid, arginine, lysine, tyrosine, and / or serine residues);1.3 Composition 1.2, wherein the N-terminal amino groups and / or internal lysine or arginine residue side chains are modified, e.g., acylated (e.g., acetylated);1.4 Composition 1.2, or 1.3, wherein the C-terminal amino groups and / or internal glutamic acid side chains are modified, e.g., esterified or amidated (e.g., as a primary amide or N-alkyl amide);1.5 Composition 1.2, 1.3, or 1.4, wherein the internal tyrosine and / or serine residues are modified, e.g., acylated (e.g., acetylated), or etherized (e.g., as methyl ethers);1.6 Composition 1.4, wherein the polypeptide is a polypeptide according to SEQ ID NO. 1, modified with an N-terminal acetyl group and a C-terminal primary amide;1.7 Composition 1, wherein the polypeptide is a polypeptide comprising the polypeptide according to any one of embodiments 1.1 -1.6 with additional amino acid residues inserted before or after the residues of SEQ ID NO.l;1.8 Composition 1, wherein the polypeptide is a fragment of the polypeptide according to SEQ ID NO. 1, having one or more amino acid residues omitted from the sequence (i.e., a peptide fragment of SEQ ID NO. 1);1.9 Composition 1.8, wherein the polypeptide has any 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 amino acids of SEQ ID NO.l omitted;Composition 1.8 or 1.9, wherein the polypeptide comprises at least the sequences IAWYK (SEQ ID NO. 25), WIAWYKL (SEQ ID NO. 24), or YWIAWYKLL (SEQ ID NO. 22); Composition 1.8, 1.9, or 1.10, wherein the polypeptide comprises at least 14, 15, 16, 17, or 18 of the amino acids of SEQ ID NO. 1, preferably wherein the polypeptide comprises at least 14, 15, 16, 17, or 18 consecutive amino acids of the amino acids of SEQ ID NO. 1; Composition, 1 , or any of 1.8- 1.11 , wherein the polypeptide is a polypeptide having one or more amino acids substituted with an alternative amino acid, such that the resulting polypeptide has at least 80% sequence identity to the polypeptide according to Composition 1, or any of 1.8-1.11, e.g., at least 85% sequence identity, or at least 90% sequence identity, or at least 95% sequence identity (e.g., as calculated by the BLASTP sequence identification and similarity tool of the NCBI Protein Blast program); Composition 1.12, where one or more neutral amino acids (e.g., glycine, alanine, isoleucine, leucine, is substituted with a different neutral amino acid (e.g., glycine, alanine, valine, leucine, isoleucine); Composition 1.12, or 1.13, wherein one or more basic amino acids (e.g., arginine, lysine) is substituted with a different basic amino acid (e.g., arginine, lysine, histidine); Composition 1.12, 1.13, or 1.14, wherein one or more acidic amino acids (e. g. , glutamic acid) is substituted with a different acidic amino acid (e.g., aspartic acid); Any of Compositions 1.11-1.15, wherein one or more hydroxylic amino acids (e.g., serine, tyrosine) is substituted with a different hydroxylic amino acid (e.g., serine, tyrosine, threonine); Any of Compositions 1.11-1.16, wherein one or more aromatic amino acids (e.g., phenylalanine, tryptophan, tyrosine) is substituted with a different aromatic amino acid (e.g., phenylalanine, tryptophan, tyrosine); Any of Compositions 1.11-1.17, wherein one or more asparagine or glutamine residues is substituted with a different residue (e.g., glutamine, or asparagine);1.19 Any of Compositions 1.11-1.18, comprising any of the following substitutions: R to A, R to E, Q to A, Y to A, F to A, F to Y, W to A, I to A, W to Y, Y to A, K to A, K to E, L to I, A to L, N to S, N to A, S to N, S to A, K to A, or K to E;1.20 Any of Compositions 1.11-1.18, wherein the polypeptide is a polypeptide comprising the polypeptide according to any one of embodiments 1.11-1.18 with additional amino acid residues inserted before or after the indicated residues;1.21 Composition 1.20, wherein the polypeptide comprises two or more sequences according to any one of embodiments 1.11-1.18, separated by polyglutamine sequences of 3 to 10 residues (e.g., 5 residues); 1.22 Composition 1.21, wherein the polypeptide comprises two or more sequencesIAWYK (SEQ ID NO. 25), WIAWYKL (SEQ ID NO. 24), or YWIAWYKLL (SEQ ID NO. 22), separated by polyglutamine sequences of 3 to 10 residues (e.g., 5 residues);1.23 Any of Compositions 1.11 - 1.22, wherein the polypeptide comprises a sequence as provided, or is a polypeptide having a sequence, as provided in any embodiment in the following table (sequence identity values calculated by the BLASTP sequence identification and similarity tool of the NCBI Protein Blast program):1.24 Any of Compositions 1.8-1.23, wherein the polypeptide is the polypeptide according to said embodiment, without modification (derivatization of any amino acids);1.25 Any of Compositions 1.8-1.24, wherein the polypeptide is the polypeptide according to said embodiment, wherein the polypeptide further comprises one or more modifications of the N-terminal amino group, C-terminal carboxyl group, and / or modifications of the internal amino acid amino and / or carboxyl groups and / or hydroxyl groups (e.g., of any glutamic acid, aspartic acid, arginine, lysine, histidine, tyrosine, and / or serine residues); 1.26 Composition 1.25, wherein the N-terminal amino groups and / or any internal lysine or arginine residue side chains are modified, e.g., acylated (e.g., acetylated);1.27 Composition 1.25, or 1.26, wherein the C-terminal amino groups and / or any internal glutamic acid or aspartic acid side chains are modified, e.g., esterified or amidated (e.g., as a primary amide or N-alkyl amide); 1.28 Composition 1.25, 1.26, or 1.27, wherein any internal tyrosine, threonine, and / or serine residues are modified, e.g., acylated (e.g., acetylated), or etherized (e.g., as methyl ethers);Composition 1.28, wherein the polypeptide is a polypeptide according to any of SEQ ID NO. 2 to SEQ ID NO.31, modified with an N-terminal acetyl group and a C- terminal primary amide; Composition 1, or any of 1.1-1.29, wherein the polypeptide is in free form (i.e., not a salt); Composition 1, or any of 1.1-1.29, wherein the polypeptide is in a salt form, e.g., an acetate, chloride, bromide, carbonate, phosphate, palmitate, caproate, or histidate; Composition 1 , or any of Compositions 1.1-1.31, wherein the polypeptide has a water content of less than 12%, e.g., as measured by Karl Fischer assay, e.g., less than 11%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1 %, less than 0.75% or less than 0.5%, or less than 0.25% or less than 0.15%; Composition 1, or any of Compositions 1.1-1.32, wherein the polypeptide has a mass loss on vacuum drying of less than 10%, e.g., at 0.001 mbar and 32 °C, e.g., less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1 %, or 0.5 to 10%, 0.5 to 8 %, 0.5 to 6%, 1.0 to 10 %, 1.0 to 8 %, 1.0 to 6%, 1.0 to 5%, 1.0 to 4%, 2.0 to 10%, 2.0 to 8 %, 2.0 to 6%, 2.0 to 5%, 2.5 to 10%, 2.5 to 8%, 2.5 to 6 %, 2.5 to 5%, 3.0 to 10%, 3.0 to 8 %, 3.0 to 6%, 3.0 to 5%, 4.0 to 10%, 4.0 to 8 %, 4.0 to 6 %, 4.0 to 5%, 4.0 to 5.5 %, 4.5 to 5.5%, or about 5%; Composition 1, or any of 1.1-1.33, wherein the non-aqueous liquid vehicle comprises one or more semifluorinated alkanes having the chemical formula F(CF2)n(CH2)mH, wherein n is an integer selected from 4 to 8 and m is an integer selected from 2 to 10; Composition 1 , or any of 1.1 - 1.33 , wherein the non-aqueous liquid vehicle comprises one or more semifluorinated alkanes having the chemical formula F(CF2)n(CH2)mH, wherein n is an integer selected from 4 to 8 and m is an integer selected from 4 to 8; Composition 1 , or any of 1.1 - 1.33 , wherein the non-aqueous liquid vehicle comprises one or more semifluorinated alkanes selected from the group consisting of F(CF2)4(CH2)4H, F(CF2)4(CH2)5H, F(CF2)4(CH2)6H, F(CF2)4(CH2)8H,F(CF2)6(CH2)2H, F(CF2)6(CH2)4H, F(CF2)6(CH2)6H, F(CF2)6(CH2)8H, and F(CF2)6(CH2)IOH;1.37 Composition 1 , or any of 1.1 - 1.33 , wherein the non-aqueous liquid vehicle comprises one or more semifluorinated alkanes selected from the group consisting of F(CF2)4(CH2)5H, F(CF2)6(CH2)6H, F(CF2)6(CH2)8H, and F(CF2)6(CH2)IOH;1.38 Composition 1 , or any of 1.1 - 1.33 , wherein the non-aqueous liquid vehicle comprises the semifluorinated alkane F(CF2)4(CH2)sH;1.39 Composition 1 , or any of 1.1 - 1.33 , wherein the non-aqueous liquid vehicle comprises the semifluorinated alkane F(CF2)g(CH2)8H;1.40 Composition 1, or any of 1.1-1.39, wherein the composition comprises two or more semifluorinated alkanes (e.g., comprising at least F(CF2)4(CH2)sH or F(CF2)6(CH2)8H);1.41 Composition 1 , or any of 1.1 - 1.40, wherein the non-aqueous liquid vehicle comprises a linear semifluorinated alkane and a branched semifluorinated alkane of the same formula (i.e., a structural isomer), optionally wherein the branched isomer is present in an amount of 0.1-5% by weight, or 0.1 to 1%, 0.1 to 0.5%, or 0.2 to 0.5% by weight of the linear isomer;1.42 Composition 1.41, wherein the branched semifluorinated alkane is the 2- perfluoroalkyl isomer of the linear (1 -perfluoroalkyl) semifluorinated alkane;1.43 Composition 1 , or any of 1.1 - 1.42, wherein the composition comprises the two semifluorinated alkanes F(CF2)4(CH2)sH and F(CF2)4(CH)(CH3)(CH2)3H;1.44 Composition 1, or any of 1.1-1.42, wherein the composition comprises the two semifluorinated alkanes F(CF2)g(CH2)8H and F(CF2)6(CH)(CH3)(CH2)sH;1.45 Composition 1, or any of 1.1-1.42, wherein the composition comprises a single semifluorinated alkane (e.g., F(CF2)4(CH2)sH or F(CF2)e(CH2)8H);1.46 Composition 1, or any of 1.1-1.45, wherein the composition is a dispersion;1.47 Composition 1, or any of 1.1-1.45, wherein the composition is a suspension;1.48 Composition 1.47, wherein the composition comprises particles comprising the polypeptide suspended in the non-aqueous liquid vehicle;1.49 Composition 1.48, wherein the particles have a particle size characterized by an X90 of less than 50 microns, less than 40 microns, less than 30 microns, less than 25microns, less than 20 microns, or less than 15 microns, or less than 10 microns, e.g., 1 to 50 microns, 1 to 40 microns, 1 to 30 microns, 1 to 25 microns, 1 to 20 microns, 1 to 15 microns, 1 to 10 microns, 5 to 50 microns, 5 to 40 microns, 5 to 30 microns, 5 to 25 microns, 5 to 20 microns, 5 to 15 microns, 5 to 10 microns, 10 to 50 microns, 10 to 40 microns, 10 to 30 microns, 10 to 25 microns, 10 to 20 microns, 10 to 15 microns, 20 to 50 microns, 20 to 40 microns, 20 to 30 microns, or 20 to 25 microns, or about 15 microns, or about 17 microns, e.g. as determined by laser diffraction (e.g., at a wavelength of 632.8 nm and / or measurement range of 0.25-87.5 gm), and optionally wherein the particles have not been subject to ultrasound sonifi cation;1.50 Composition 1.48 or 1.49, wherein the particles have a particle size characterized by an X50 of less than 50 microns, less than 40 microns, less than 30 microns, less than 25 microns, less than 20 microns, less than 15 microns, less than 10 microns, less than 7.5 microns, or less than 5 microns, e.g., 1 to 50 microns, 1 to 40 microns, 1 to 30 microns, 1 to 25 microns, 1 to 20 microns, 1 to 15 microns, 1 to 10 microns, 3 to 50 microns, 3 to 40 microns, 3 to 30 microns, 3 to 25 microns, 3 to 20 microns, 3 to 15 microns, 3 to 10 microns, 3 to 8 microns, 5 to 50 microns, 5 to 40 microns, 5 to30 microns, 5 to 25 microns, 5 to 20 microns, 5 to 15 microns, 5 to 10 microns, 4 to 8 microns, 4 to 6 microns, or about 5 microns, e.g. as determined by laser diffraction (e.g., at a wavelength of 632.8 nm and / or measurement range of 0.25-87.5 pm); and optionally wherein the particles have not been subject to ultrasound sonifi cation;1.51 Any of Compositions 1.48-1.50, wherein 90% of the particles have a particle size characterized by a range of 1 to 100 microns, 1 to 50 microns, 1 to 40 microns, 1 to 30 microns, 1 to 20 microns, 1 to 25 microns, 1 to 15 microns, 1 to 10 microns, 2 to 100 microns, 2 to 50 microns, 2 to 40 microns, 2 to 30 microns, 2 to 20 microns, 2 to 25 microns, 2 to 15 microns, 2 to 10 microns, 3 to 40 microns, 3 to 30 microns, 3 to 25 microns, 3 to 20 microns, 3 to 15 microns, 3 to 10 microns, 4 to 50 microns, 4 to 40 microns, 4 to 30 microns, 4 to 25 microns, or 4 to 20 microns, 4 to 15 microns, 4 to 10 microns, e.g. as determined by laser diffraction (e.g., at a wavelength of 632.8 nm and / or measurement range of 0.25-87.5 pm), and optionally wherein the particles have not been subject to ultrasound sonifi cation;1.52 Any of Compositions 1.48-1.51, wherein the particles have a mean particle diameter of 1 to 100 microns, 1 to 50 microns, 1 to 40 microns, 1 to 30 microns, 1 to 20 microns, 1 to 15 microns, 1 to 10 microns, 2 to 100 microns, 2 to 50 microns, 2 to40 microns, 2 to 30 microns, 2 to 20 microns, 2 to 15 microns, 2 to 10 microns, 3 to40 microns, 3 to 30 microns, 3 to 20 microns, 3 to 15 microns, 3 to 10 microns, 4 to50 microns, 4 to 40 microns, 4 to 30 microns, 4 to 25 microns, or 4 to 20 microns, 4 to 15 microns, 4 to 10 microns, e.g. as determined by laser diffraction (e.g., at a wavelength of 632.8 nm and / or measurement range of 0.25-87.5 pm), and optionally wherein the particles have not been subject to ultrasound sonification;1.53 Any of Compositions 1.48-1.52, wherein the particles have a water content of less than 12%, e.g., as measured by Karl Fischer assay, e.g., less than 11%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1 %, less than 0.75%, less than 0.5%, less than 0.25%, or less than 0.15%;1.54 Any of Compositions 1.48-1.53, wherein the particles have a mass loss on vacuum drying of less than 10%, e.g., at 0.001 mbar and 32 °C, e.g., less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1 %, or 0.5 to 10%, 0.5 to 8 %, 0.5 to 6%, 1.0 to 10 %, 1.0 to 8 %, 1.0 to 6%, 1.0 to 5%, 1.0 to 4%, 2.0 to 10%, 2.0 to 8 %, 2.0 to 6%, 2.0 to 5%, 2.5 to 10%, 2.5 to 8%, 2.5 to 6 %, 2.5 to 5%, 3.0 to 10%, 3.0 to 8 %, 3.0 to 6%, 3.0 to 5%, 4.0 to 10%, 4.0 to 8 %, 4.0 to 6 %, 4.0 to 5%, 4.0 to 5.5 %, 4.5 to 5.5%, or about 5%;1.55 Composition 1 , or any of 1.1 -1.54, wherein the composition comprises the polypeptide at a concentration of about 0.1 to 50 mg / mL, e.g., 0.1 to 25 mg / mL, 0.1 to 20 mg / mL, 0.1 to 15 mg / mL, 0.1 to 10 mg / mL, 0.1 to 8 mg / mL, 0.1 to 6 mg / mL, 0.1 to 5 mg / mL, 0.1 to 4 mg / mL, 0.1 to 3 mg / mL, 0.1 to 2 mg / mL, 0.1 to 1.5 mg / mL, 0.1 to 1.25 mg / mL, 0.5 to 25 mg / mL, 0.5 to 15 mg / mL, 0.5 to 10 mg / mL, 0.5 to 5 mg / mL, 0.5 to 4 mg / mL, 0.5 to 3 mg / mL, 0.5 to 2 mg / mL, 0.5 to 1.5 mg / mL, 0.5 to 1.25 mg / mL, 0.8 to 1.2 mg / mL, 0.9 to 1.1 mg / mL, 1 to 20 mg / mL, 1 to 15 mg / mL, 1 to 10 mg / mL, 1 to 5 mg / ml, 2 to 8 mg / mL, 3 to 7 mg / mL, 4 to 6 mg / mL, 5 to 15mg / mL, 5 to 10 mg / mL, or about 0.5 mg / ml, or about 1 mg / mL, or about 4 mg / ml, or about 5 mg / mL, or about 10 mg / mL;1.56 Composition 1, or any of 1.1-1.55, wherein the composition further comprises one or more excipients selected from organic co-solvents (e.g., ethanol), stabilizing agents, acids, bases, salts (inorganic or organic), buffers, pH-modifying agents, chelating agents, surfactants, antioxidants, stabilizers, synergists, coloring agents, thickening agents, and preservatives; or other components that are miscible or soluble with the semifluorinated alkane;1.57 Composition 1 , or any of 1.1 -1.56, wherein the non-aqueous liquid vehicle comprises the one or more semifluorinated alkanes in an amount of at least 70 wt%, 75 wt%, 85 wt%, 90 wt%, 92%, 94%, 95 wt%, 96%, 97%, 98%, or 99 wt% with respect to the total weight of the liquid vehicle, up to a maximum of 99.9 wt%, e.g., wherein the non-aqueous liquid vehicle comprises the one or more semifluorinated alkanes in an amount 70-99%, 75-99%, 85-99%, 90-99%, 92-99%, 93-99%, 94-99%, 95-99%, 96-99%, or 97-99%, or 98-99%, by weight of the liquid vehicle;1.58 Composition 1 , or any of 1.1 -1.57, wherein the composition comprises the one or more semifluorinated alkanes in an amount of at least 70 wt%, 75 wt%, 85 wt%, 90 wt%, 92%, 94%, 95 wt%, 96%, 97%, 98%, or 99 wt% with respect to the total weight of the composition, up to a maximum of 99.9 wt%, e.g., wherein the composition comprises the one or more semifluorinated alkanes in an amount 70-99%, 75-99%, 85-99%, 90-99%, 92-99%, 93-99%, 94-99%, 95-99%, 96-99%, or 97-99%, or 98- 99%, by weight of the composition;1.59 Composition 1 , or any of 1.1 -1.58, wherein the particles further comprise one or more excipients, for example, selected from stabilizing agents, stabilizing agents, acids, bases, salts (inorganic or organic), buffers, pH-modifying agents, chelating agents, surfactants, antioxidants, stabilizers, synergists, coloring agents, thickening agents, and preservatives;1.60 Composition 1 , or any of 1.1 -1.59, wherein the composition is free or substantially free of water;1.61 Composition 1, or any of 1.1-1.60, wherein the composition is free or substantially free of surfactants;1.62 Composition 1 , or any of 1.1 - 1.61 , wherein the composition is free or substantially free of preservatives;1.63 Composition 1 , or any of 1.1 - 1.62, wherein the composition comprises the polypeptide according to SEQ ID NO. 1, modified with an N-terminal acetyl group and a C-terminal primary amide, suspended in the non-aqueous vehicle comprising the semifluorinated alkane F(CF2)6(CH2)sH, and optionally F(CF2)6(CH)(CH3)(CH2)eH, wherein the composition comprises the polypeptide at a concentration of about 0.1 to 5 mg / mL (e.g., about 1 mg / mL or about 5 mg / mL), and the semifluorinated alkane(s) in an amount of 95-99% by weight of the liquid vehicle;1.64 Composition 1, or any of 1.1-1.63, wherein the composition consists of or consists essentially of the polypeptide suspended in the non-aqueous vehicle and optionally one or more excipients, wherein the non-aqueous vehicle consists of the one or more semifluorinated alkanes;1.65 Composition 1, or any of 1.1-1.63, wherein the composition consists of or consists essentially of particles comprising the polypeptide suspended in the nonaqueous vehicle and optionally one or more excipients, wherein the non-aqueous vehicle consists of the one or more semifluorinated alkanes;1.66 Composition 1.64 or 1.65, wherein the polypeptide is the polypeptide according to SEQ ID NO. 1, modified with an N-terminal acetyl group and a C-terminal primary amide;1.67 Composition 1.64 or 1.65, wherein the non-aqueous vehicle consists of the semifluorinated alkane F(CF2)6(CH2)sH, and optionally F(CF2)6(CH)(CH3)(CH2)6H;1.68 Any of Compositions 1.64-1.67, wherein the polypeptide is present at a concentration of about 0.1 to 5 mg / mL (e.g., about 1 mg / mL or about 5 mg / mL), and the semifluorinated alkane(s) is present in an amount of 95-99% by weight of the liquid vehicle;1.69 Composition 1, or any of 1.1-1.68, wherein the composition is physically stable, e.g., under accelerated aging conditions of 25 °C / 60% relative humidity, e.g., for 2 weeks, 4 weeks, 2 months, 3 months, or 6 months;1.70 Composition 1.69, wherein the X50 and / or X90 parameters of the particle size distribution of the suspension changes by less than 20% or less than 15% or less than10% or less than 5%, e.g. as determined by laser diffraction (e.g., at a wavelength of 632.8 nm and / or measurement range of 0.25-87.5 gm), under accelerated aging conditions of 25 °C / 60% relative humidity, e.g., for 2 weeks, 4 weeks, 2 months, 3 months, or 6 months;1.71 Composition 1, or any of 1.1-1.70, wherein the composition is chemically stable, e.g., under accelerated aging conditions of 25 °C / 60% relative humidity, e.g., for 2 weeks, 4 weeks, 2 months, 3 months, or 6 months;1.72 Composition 1.71, wherein the concentration of the polypeptide changes by less than 15%, or less than 10%, or less than 8%, or less than 6%, or less than 5%, or less than 4%, or less than 3%, under accelerated aging conditions of 25 °C / 60% relative humidity, e.g., for 2 weeks, 4 weeks, 2 months, 3 months, or 6 months;1.73 Composition 1.71 or 1.72, wherein during storage, the composition forms less than 10% of any one or more truncated degradation products of the polypeptide, e.g., less than less than 8%, or less than 6%, or less than 5%, or less than 4%, or less than 3%, less than 2%, less than 1%, less than 0.5%, or less than 0.1%, e.g., under accelerated aging conditions of 25 °C / 60% relative humidity, e.g., for 2 weeks, 4 weeks, 2 months, 3 months, or 6 months, for example, wherein the polypeptide comprises the polypeptide according to SEQ ID NO. 1, modified with an N-terminal acetyl group and a C-terminal primary amide, and wherein said truncated degradation products include one or more of the polypeptides according to any of SEQ ID NO. 6, 18-21, 23, 24, and 28.

[0047] In some embodiments, the polypeptide of the Composition 1 et seq. is selected from the following, wherein bold indicates amino acids having sequence identity to SEQ ID NO. 1 (sequence identity values calculated by the BLASTP sequence identification and similarity tool of the NCBI Protein Blast program):Without being bound by theory, it is believed that one or more of SEQ ID. NO. 2 to NO. 31 may be physiologically active, e.g., comparable to SEQ ID NO. 1, but may have increased stability towards enzymatic and / or non-enzymatic hydrolysis, either during storage or during application to a patient (e.g., during transit of the ocular space to the retina).

[0048] In a second aspect, the present disclosure provides a pharmaceutical composition (e.g.,Composition 1, or any of 1.1 et seq.) comprising a polypeptide, or a pharmaceutically acceptable salt thereof, dispersed, or suspended in a non-aqueous liquid vehicle comprising one or more semifluorinated alkanes, wherein the polypeptide is a polypeptide according to SEQ ID NO. 1, or an analog and / or derivative thereof, for use as a medicament or for use in a method of treating orpreventing a posterior eye disorder (e.g., diabetic retinopathy). Further embodiments of the present disclosure provide as follows:1.74 Composition 1, or any of 1.1-1.73, for use as a medicament;1.75 Composition 1, or any of 1.1-1.74, for use as a medicament in ophthalmology;1.76 Composition 1, or any of 1.1-1.75, for use as a topical ophthalmic medicament;1.77 Composition 1, or any of 1.1-1.76, for use in a method of treating or preventing a posterior eye disorder (e.g., diabetic retinopathy);1.78 Composition 1.77, for use wherein the posterior eye disorder is selected from glaucoma, ocular hypertension, age-related macular degeneration, diabetic retinopathy, retinal vein occlusion, retinal artery occlusion, retinal ischemia, ischemic optic neuropathy, macular edema, uveitis, endophthalmitis, and retinal detachment;1.79 Composition 1.77, for use wherein the posterior eye disorder is retinal ischemia;1.80 Composition 1.77, for use wherein the posterior eye disorder is diabetic retinopathy and / or diabetic macular edema;1.81 Composition 1.80, for use wherein the diabetic retinopathy and / or diabetic macular edema is consequent to Type 1 diabetes;1.82 Composition 1.80, for use wherein the diabetic retinopathy and / or diabetic macular edema is consequent to Type 2 diabetes;1.83 Composition 1.80, for use wherein the diabetic retinopathy and / or diabetic macular edema is consequent to gestational diabetes;1.84 Any of Compositions 1.78-1.83, for use wherein the diabetic retinopathy is nonproliferative diabetic retinopathy, e.g., mild, moderate, or severe non-proliferative diabetic retinopathy (e.g., according to the AAO criteria);1.85 Any of Compositions 1.78-1.83, for use wherein the diabetic retinopathy is proliferative diabetic retinopathy;1.86 Composition 1.85, for use wherein the proliferative diabetic retinopathy is associated with neovascular glaucoma;1.87 Any of Compositions 1.78-1.86, for use wherein the diabetic macular edema is mild macular edema, moderate macular edema, or severe macular edema (e.g., according to the AAO criteria), optionally assessed by optical coherence tomography;1.88 Composition 1 , or any of 1.1 -1.76, for use wherein the composition or medicament is for use in delivering the polypeptide, or a metabolite thereof, to the eye, e.g., to the intraocular tissues, posterior eye segment, retinal tissues, and / or optic nerve;1.89 Composition 1.88, for use wherein the polypeptide is delivered to the choroid or retina;1.90 Composition 1, or any of 1.1-1.76, wherein the composition or medicament is for use in protecting the retinal ganglion cells and / or the optic nerve cells, e.g., against ischemia, ischemic injury, or ischemia-induced injury, such as maintaining normal thickness and or morphology in the inner nuclear layer (INL) and / or outer nuclear layer (ONL) of the retina;1.91 Composition 1, or any of 1.1-1.76, wherein the composition or medicament is for use in inducing nitric oxide synthase or increasing nitric oxide production in the tissues of the posterior eye (e.g., the retina, macula, and / or optic nerve);1.92 Any of Compositions 1.74-1.91, for use wherein the composition or medicament is administered to a patient suffering from Type 1 diabetes, Type 2 diabetes, or gestational diabetes;1.93 Composition 1.92, for use wherein the patient is characterized as having one or more ocular abnormalities selected from microaneurysms, cotton wool spots, hemorrhages, hard exudates, intraretinal microvascular abnormalities, and abnormal- looking retinal veins;1.94 Any of Compositions 1.74-1.93, for use wherein the composition or medicament is administered to a patient suffering a posterior eye disorder selected from glaucoma, ocular hypertension, age-related macular degeneration, diabetic retinopathy, retinal vein occlusion, retinal artery occlusion, retinal ischemia, ischemic optic neuropathy, macular edema, uveitis, endophthalmitis, and retinal detachment;1.95 Composition 1.94, for use wherein the posterior eye disorder is retinal ischemia;1.96 Composition 1.94, for use wherein the posterior eye disorder is diabetic retinopathy and / or diabetic macular edema;1.97 Composition 1.96, for use wherein the diabetic retinopathy and / or diabetic macular edema is consequent to Type 1 diabetes;1.98 Composition 1.96, for use wherein the diabetic retinopathy and / or diabetic macular edema is consequent to Type 2 diabetes;1.99 Composition 1.96, for use wherein the diabetic retinopathy and / or diabetic macular edema is consequent to gestational diabetes;1.100 Any of Compositions 1.94-1.99, for use wherein the diabetic retinopathy is nonproliferative diabetic retinopathy, e.g., mild, moderate, or severe non-proliferative diabetic retinopathy (e.g., according to the AAO criteria);1.101 Any of Compositions 1.94-1.99, for use wherein the diabetic retinopathy is proliferative diabetic retinopathy;1.102 Composition 1.101 , for use wherein the proliferative diabetic retinopathy is associated with neovascular glaucoma;1.103 Any of Compositions 1.94-1.99, for use wherein the diabetic macular edema is mild macular edema, moderate macular edema, or severe macular edema (e.g., according to the AAO criteria), optionally assessed by optical coherence tomography;1.104 Any of Compositions 1.92-1.103, for use wherein the patient has had Type 1 diabetes for at least 5 years, or has been diagnosed with Type 1 diabetes for at least 5 years;1.105 Any of Compositions 1.92-1.103, for use wherein the patient has had Type 1 diabetes for at least 10 years, or has been diagnosed with Type 1 diabetes for at least 10 years;1.106 Any of Compositions 1.92-1.103, for use wherein the patient has had Type 1 diabetes for at least 15 years, or has been diagnosed with Type 1 diabetes for at least 15 years;1.107 Any of Compositions 1.92-1.106, for use wherein the patient has elevated serum HbAlc (e.g., wherein the serum HbAlc level is above 48mmol / mol);1.108 Any of Compositions 1.92-1.107, for use wherein the patient has kidney disease, abnormal blood lipid levels, or abnormal body mass index;1.109 Any of Compositions 1.92-1.108, for use wherein the patient is a smoker (e.g., has a history of smoking of at least 5 years, or at least 10 years);1.110 Any of Compositions 1.92-1.109, for use wherein the patient has a mutation in the VEGFC gene;1.111 Any of Compositions 1.92-1.109, for use wherein the patient has not responded to treatment with topical ophthalmic or intravitreal corticosteroids, e.g., intravitreal triamcinolone;1.112 Any of Compositions 1.74-1.111, for use wherein the composition is administered topically to the surface of the eye (e.g., the cornea and / or conjunctiva);1.113 Composition 1.112, for use wherein the composition is administered in the form of liquid drops having a volume of 8 to 15 microliters, or 9 to 11 microliters, or 10 to 12 microliters, or about 11 microliters, or about 10 microliters;1.114 Any of Compositions 1.74-1.113, for use wherein the composition increases, normalizes, or maintains retinal layer thickness, e.g., in a patient suffering from retinal ischemia, e.g., in the inner nuclear layer and / or the outer nuclear layer of the retina;1.115 Any of Compositions 1.74-1.114, for use wherein the composition increases, normalizes, or maintains retinal ganglion cell density in the ganglion cell layer of the retina, e.g., in a patient suffering from retinal ischemia.

[0049] In a third aspect, the present disclosure provides a method (Method 2) of treating or preventing a posterior eye disorder (e.g., diabetic retinopathy), comprising administering to a patient in need thereof a pharmaceutical composition (e.g., Composition 1, or any of 1.1 et seq.) comprising a polypeptide, or a pharmaceutically acceptable salt thereof, dispersed, or suspended in a non-aqueous liquid vehicle comprising one or more semifluorinated alkanes, wherein the polypeptide is a polypeptide according to SEQ ID NO. 1, or an analog and / or derivative thereof. Further embodiments of the present disclosure provide as follows:2.1 Method 2, wherein pharmaceutical composition is any one of Compositions 1.1 to 1.73;2.2 Method 2 or 2.1, wherein the posterior eye disorder is selected from glaucoma, ocular hypertension, age-related macular degeneration, diabetic retinopathy, retinal vein occlusion, retinal artery occlusion, retinal ischemia, ischemic optic neuropathy, macular edema, uveitis, endophthalmitis, and retinal detachment;2.3 Method 2 or 2.1, wherein the posterior eye disorder is retinal ischemia;2.4 Method 2 or 2.1, wherein the posterior eye disorder is diabetic retinopathy and / or diabetic macular edema;2.5 Method 2.4, wherein the diabetic retinopathy and / or diabetic macular edema is consequent to Type 1 diabetes;2.6 Method 2.4, wherein the diabetic retinopathy and / or diabetic macular edema is consequent to Type 2 diabetes;2.7 Method 2.4, wherein the diabetic retinopathy and / or diabetic macular edema is consequent to gestational diabetes;2.8 Any of Method 2.2-2.7, wherein the diabetic retinopathy is non-proliferative diabetic retinopathy, e.g., mild, moderate, or severe non-proliferative diabetic retinopathy (e.g., according to the AAO criteria);2.9 Any of Method 2.2-2.7, wherein the diabetic retinopathy is proliferative diabetic retinopathy;2.10 Method 2.9, wherein the proliferative diabetic retinopathy is associated with neovascular glaucoma;2.11 Any of Method 2.2-2.10, wherein the diabetic macular edema is mild macular edema, moderate macular edema, or severe macular edema (e.g., according to the AAO criteria), optionally assessed by optical coherence tomography;2.12 Method 2, or any of 2.1 -2.11 , wherein the method delivers the polypeptide, or a metabolite thereof, to the eye, e.g., to the intraocular tissues, posterior eye segment, retinal tissues, and / or optic nerve;2.13 Method 2.12, wherein the polypeptide is delivered to the choroid or retina;2.14 Method 2, or any of 2.1-2.13, wherein the method protects the retinal ganglion cells and / or the optic nerve cells, e.g., against ischemia, ischemic injury, or ischemia- induced injury, such as maintaining normal thickness and or morphology in the inner nuclear layer (INL) and / or outer nuclear layer (ONL) of the retina;2.15 Method 2, or any of 2.1 -2.14, wherein the patient is suffering from Type 1 diabetes, Type 2 diabetes, or gestational diabetes;2.16 Method 2.15, wherein the patient is characterized as having one or more ocular abnormalities selected from microaneurysms, cotton wool spots, hemorrhages, hard exudates, intraretinal microvascular abnormalities, and abnormal-looking retinal veins;2.17 Method 2, or any of 2.1-2.16, wherein the patient has had Type 1 diabetes for at least 5 years, or has been diagnosed with Type 1 diabetes for at least 5 years;2.18 Method 2, or any of 2.1 -2.16, wherein the patient has had Type 1 diabetes for at least 10 years, or has been diagnosed with Type 1 diabetes for at least 10 years;2.19 Method 2, or any of 2.1-2.16, wherein the patient has had Type 1 diabetes for at least 15 years, or has been diagnosed with Type 1 diabetes for at least 15 years;2.20 Method 2, or any of 2.1-2.19, wherein the patient has elevated serum HbAlc (e.g., wherein the serum HbAlc level is above 48mmol / mol);2.21 Method 2, or any of 2.1-2.20, wherein the patient has kidney disease, abnormal blood lipid levels, or abnormal body mass index;2.22 Method 2, or any of 2.1-2.21, wherein the patient is a smoker (e.g., has a history of smoking of at least 5 years, or at least 10 years);2.23 Method 2, or any of 2.1-2.22, wherein the patient has a mutation in the VEGFC gene;2.24 Method 2, or any of 2.1-2.23, wherein the patient has not responded to treatment with a topical ophthalmic or intravitreal corticosteroid, e.g., intravitreal triamcinolone;2.25 Method 2, or any of 2.1-2.24, wherein the composition is administered topically to the surface of the eye (e.g., the cornea and / or conjunctiva);2.26 Method 2.25, wherein the composition is administered in the form of liquid drops having a volume of 8 to 15 microliters, or 9 to 11 microliters, or 10 to 12 microliters, or about 10 microliters, or about 11 microliters;2.27 Method 2, or any of 2.1-2.26, wherein the patient is concurrently undergoing treatment with a topical ophthalmic or intravitreal corticosteroid, e.g., intravitreal triamcinolone;2.28 Method 2, or any of 2.1-2.27, wherein the composition increases, normalizes, or maintains retinal layer thickness, e.g., in a patient suffering from retinal ischemia, e.g., in the inner nuclear layer and / or the outer nuclear layer of the retina;2.29 Method 2, or any of 2.1-2.28, wherein the composition increases, normalizes, or maintains retinal ganglion cell density in the ganglion cell layer of the retina, e.g., in a patient suffering from retinal ischemia.

[0050] In a fourth aspect, the present disclosure provides a method (Method 3) of inducing nitric oxide synthase or increasing nitric oxide production in the tissues of the posterior eye (e.g., the retina, macula, and / or optic nerve), comprising administering to a patient in need thereof a pharmaceutical composition (e.g., Composition 1, or any of 1.1 et seq.) comprising a polypeptide, or a pharmaceutically acceptable salt thereof, dispersed, or suspended in a nonaqueous liquid vehicle comprising one or more semifluorinated alkanes, wherein the polypeptide is a polypeptide according to SEQ ID NO. 1, or an analog and / or derivative thereof. Further embodiments of the present disclosure provide as follows:3.1 Method 3, wherein pharmaceutical composition is any one of Compositions 1.1 to 1.73;3.2 Method 3 or 3.1, wherein the method induces nitric oxide synthase or increases nitric oxide production in retina, macula, choroid, optic nerve, and / or retinal ganglion cells;3.3 Method 1, or any of 3.1-3.2, wherein the patient suffers from a posterior eye disorder;3.4 Method 3.3, wherein the posterior eye disorder is selected from glaucoma, ocular hypertension, age-related macular degeneration, diabetic retinopathy, retinal vein occlusion, retinal artery occlusion, retinal ischemia, ischemic optic neuropathy, macular edema, uveitis, endophthalmitis, and retinal detachment;3.5 Method 3.3, wherein the posterior eye disorder is retinal ischemia;3.6 Method 3.3, wherein the posterior eye disorder is diabetic retinopathy and / or diabetic macular edema;3.7 Method 3.6, wherein the diabetic retinopathy and / or diabetic macular edema is consequent to Type 1 diabetes;3.8 Method 3.6, wherein the diabetic retinopathy and / or diabetic macular edema is consequent to Type 2 diabetes;3.9 Method 3.6, wherein the diabetic retinopathy and / or diabetic macular edema is consequent to gestational diabetes;3.10 Any of Methods 3.4-3.9, wherein the diabetic retinopathy is non-proliferative diabetic retinopathy, e.g., mild, moderate, or severe non-proliferative diabetic retinopathy (e.g., according to the AAO criteria);3.11 Any of Method 3.4-3.9, wherein the diabetic retinopathy is proliferative diabetic retinopathy;Method 3.11, wherein the proliferative diabetic retinopathy is associated with neovascular glaucoma; Any of Methods 3.4-3.12, wherein the diabetic macular edema is mild macular edema, moderate macular edema, or severe macular edema (e.g., according to the AAO criteria), optionally assessed by optical coherence tomography; Method 3, or any of 3.1-3.13, wherein the method delivers the polypeptide, or a metabolite thereof, to the eye, e.g., to the intraocular tissues, posterior eye segment, retinal tissues, and / or optic nerve; Method 3.14, wherein the polypeptide is delivered to the choroid or retina; Method 3, or any of 3.1-3.15, wherein the patient is suffering from Type 1 diabetes, Type 2 diabetes, or gestational diabetes; Method 3.16, wherein the patient is characterized as having one or more ocular abnormalities selected from microaneurysms, cotton wool spots, hemorrhages, hard exudates, intraretinal microvascular abnormalities, and abnormal-looking retinal veins; Method 3, or any of 3.1-3.17, wherein the patient has had Type 1 diabetes for at least 5 years, or has been diagnosed with Type 1 diabetes for at least 5 years; Method 3, or any of 3.1-3.17, wherein the patient has had Type 1 diabetes for at least 10 years, or has been diagnosed with Type 1 diabetes for at least 10 years; Method 3, or any of 3.1-3.17, wherein the patient has had Type 1 diabetes for at least 15 years, or has been diagnosed with Type 1 diabetes for at least 15 years; Method 3, or any of 3.1-3.20, wherein the patient has elevated serum HbAlc (e.g., wherein the serum HbAlc level is above 48mmol / mol); Method 3, or any of 3.1-3.21, wherein the patient has kidney disease, abnormal blood lipid levels, or abnormal body mass index; Method 3, or any of 3.1-3.22, wherein the patient is a smoker (e.g., has a history of smoking of at least 5 years, or at least 10 years); Method 3, or any of 3.1-3.23, wherein the patient has a mutation in the VEGFC gene;3.25 Method 3, or any of 3.1-3.24, wherein the patient has not responded to treatment with a topical ophthalmic or intravitreal corticosteroid, e.g., intravitreal triamcinolone;3.26 Method 3, or any of 3.1-3.25, wherein the composition is administered topically to the surface of the eye (e.g., the cornea and / or conjunctiva);3.27 Method 3.26, wherein the composition is administered in the form of liquid drops having a volume of 8 to 15 microliters, or 9 to 11 microliters, or 10 to 12 microliters or about 10 microliters, or about 11 microliters;3.28 Method 3, or any of 3.1-3.27, wherein the patient is concurrently undergoing treatment with a topical ophthalmic or intravitreal corticosteroid, e.g., intravitreal triamcinolone;3.29 Method 3, or any of 3.1-3.28, wherein the composition increases, normalizes, or maintains retinal layer thickness, e.g., in a patient suffering from retinal ischemia, e.g., in the inner nuclear layer and / or the outer nuclear layer of the retina;3.30 Method 3, or any of 3.1-3.29, wherein the composition increases, normalizes, or maintains retinal ganglion cell density in the ganglion cell layer of the retina, e.g., in a patient suffering from retinal ischemia.

[0051] In a related aspect to the second, third and fourth aspects of the present disclosure, the present disclosure also provides the use of a composition, e.g. Composition 1, or any of 1.1 et seq., in the manufacture or the preparation of a medicament, e.g. a medicament for use according to any of the uses or methods as described herein above e.g. uses of the Composition of any of 1.72 to 1.115, or Method 2, or any of 2.1 to 2.29, or Method 3, or any of 3.1 to 3.30.

[0052] In a fifth aspect, the present disclosure provides a kit comprising a pharmaceutical composition (e.g., Composition 1 or any of 1.1 et seq.) comprising a polypeptide, or a pharmaceutically acceptable salt thereof, dispersed, or suspended in a non-aqueous liquid vehicle comprising one or more semifluorinated alkanes, wherein the polypeptide is a polypeptide according to SEQ ID NO. 1, or an analog and / or derivative thereof, and a container for holding the pharmaceutical composition and a drop dispenser for administering the composition.Optionally, the kit further comprises instructions for use of said composition as a medicament or for use in a method of treating or preventing a posterior eye disorder (e.g., diabetic retinopathy) or for use in a method of inducing nitric oxide synthase or increasing nitric oxide production inthe tissues of the posterior eye, as disclosed herein above. Preferably, the kit comprises a drop dispenser that is adapted for topical administration of liquid drops having a volume of 8 to 15 microliters, or 9 to 11 microliters, or 10 to 12 microliters, or about 10 microliters, or about 11 microliters, to the eye of a patient.

[0053] As used herein, the term “polypeptide” only refers to a single polymer chain, whereas the expression “protein” may also refer to two or more polypeptide chains that are linked to each other by non-covalent bonds, such as disulfide bonds.

[0054] Those that are skilled in the art acknowledge that certain modifications may be made on peptides such as those described in the present disclosure, causing small or no alterations to the properties of the said peptides. Therefore, peptides related to those demonstrated herein include analogues and / or derivatives that retain some or all of the therapeutic activity of the original peptides. In this context, the term “analog” indicates variants obtained by substitutions, deletions or additions of amino acids to the peptides described herein; while “derivative” indicates variants containing chemical modifications on the primary sequence of the peptides described herein and / or their analogues. In certain aspects, such variants may evidence improvements in at least one of the therapeutic activities of the peptides. Additionally, the peptides of the present description may be comprised of L-amino acids, D-amino acids or a combination of both in any ratio. In this context, a polypeptide with an amino acid sequence selected from SEQ ID. NO. 2 through SEQ ID NO.31 is understood as being an analog and / or a derivative of the polypeptide with amino acid sequence SEQ ID NO: 1.

[0055] Desirable amino acid substitutions can be determined by those skilled in the art using routine methodologies. Natural amino acids may be classified in terms of the side chains properties of the as: nonpolar (glycine (Gly), alanine (Ala), valine (Vai), leucine (Leu), isoleucine (He), methionine (Met)); uncharged polar (cysteine (Cys), serine (Ser), threonine (Thr), proline (Pro), asparagine (Asn), glutamine (Gin); acid (aspartic acid (Asp), glutamic acid (Glu)); basic (histidine (His), lysine (Lys), arginine (Arg)); and aromatic (tryptophan (Trp), tyrosine (Tyr), phenylalanine (Phe)). Exchange of amino acid for another one of the same class generally produces variants with functional and chemical characteristics similar to those of the original peptide. This type of modification also encompasses substitutions by artificial and / or non-essential amino acid residues, including peptidomimetics and other atypical forms of amino acids that can be regularly used during the synthesis of the peptide.

[0056] Strategies for defining conservative substitutions of amino acids can be guided by the hydropathicity index of the side chains. The importance of hydropathic amino acids on the function of a polypeptide is understood by a person skilled in the art. Each amino acid has a hydropathicity index determined based on characteristics of hydrophobicity and charge. These are He (+4.5); Vai (+4.2); Leu (+3.8); Phe (+2.8); Cys (+2.5); Met (+1.9); Ala (+1.8); Gly (-0.4); Thr (-0.7); Ser (-0.8); Trp (-0.9); Tyr (-1.3); Pro (-1.6); His (-3.2); Glu (-3.5); Gin (-3.5); Asp (-3.5); Asn (-3.5); Lys (-3.9); and Arg (-4.5). Those skilled in the art understand that amino acids with similar hydropathicity indexes can be interchanged without significant loss of biological activity.

[0057] It is also known that conservative substitutions can be based on hydrophilicity. The average hydrophilicity of a polypeptide, determined by the hydrophilicity of the adjacent amino acids, is correlated with the biological properties of the compound. The natural amino acids have the following hydrophilicity values: Arg (+3.0); Lys (+3.0); Asp (+3.0+1); Glu (+3.0+1); Ser (+0.3); Asn (+0.2); Gin (+0.2); Gly (0); Thr (-0.4); Pro (-0.5+1); Ala (-0.5); His (-0.5); Cys (-1.0); Met (-1.3); Vai (-1.5); Leu (-1.8); He (-1.8); Tyr (-2.3); Phe (-2.5); and Trp (-3.4).

[0058] In some embodiments, the polypeptides of the present disclosure may be acylated, preferably acetylated, in one or more positions of the peptide chain in order to improve physicochemical, pharmacokinetic and / or pharmacodynamic characteristics. Lor example, the introduction of lipophilic acyl groups, e.g. introduction of an acetyl-group, is widely employed to increase the plasma half-life of therapeutic peptides, since they render the groups coupled thereto less susceptible to oxidations. Methods and reagents for acylation and / or acetylation of peptides are known to those familiar with the art. The modification of free amines with acyl groups, preferably with acetyl groups, is particularly useful to promote the acylation of peptides and proteins. In preferred embodiments, polypeptides disclosed herein may be acylated, preferably acetylated, at the N-terminal amine or in the side chain of one or more amino acids originally present in the sequence or inserted for the purpose of receiving the acylation or acetylation in question. Preferably, the polypeptides of the present disclosure may be acetylated at the N- terminal amine.

[0059] Similarly, the polypeptides of the present disclosure may be amidated in one or more positions of the peptide chain in order to improve physicochemical, pharmacokinetic and / or pharmacodynamic characteristics. Amidation refers to the conversion of a free carboxyl group (-COOH) to a primary amide (-CONH2) or an N-alkyl amide (-CONHR or -CONR2, wherein R is a primary 1-6 carbon alkyl chain). Unless indicated otherwise, the term “amidated” refers to the formation of a primary amide. Preferably, the polypeptides of the present disclosure may be amidated at the C-terminal carboxyl group (-COOH), more preferably the C-terminal carboxyl group may be amidated to a primary amide (-CONH2). More preferably, the polypeptides of the present disclosure may be acetylated at the N-terminal amine and amidated at the C-terminal carboxyl group to a primary amide (-CONH2).

[0060] As used herein, the term “sequence identity” refers the percentage of amino acids that are identical between two amino acid sequences being compared. The term “% sequence identity” or similar terms are intended to refer, in particular, to the percentage of amino acids which are identical in an optimal alignment between the sequences being compared. Percent sequence identity is calculated by determining the number of identical positions at which the sequences to be compared correspond, dividing this number by the number of positions compared, and multiplying this result by 100. In some embodiments, percent sequence identity of two polypeptide sequences is determined using the BLASTP algorithm, available from the website of the United States National Center for Biotechnology Information (NCBI) as part of the Protein Blast software suite. In preferred embodiments, the algorithm parameters used for BLASTP algorithm on the NCBI website include: (i) Expect Threshold set to 16; (ii) Word Size set to 2; (iii) Max matches in a query range set to 0; (iv) Matrix set to PAM30; (v) Gap Costs set to Existence: 9 Extension: 1; and (vi) conditional compositional score matrix adjustment. For example, when utilizing BLASTP for comparing the 15-mer query sequence SEQ ID NO: 2 with the 19-mer reference sequence SEQ ID NO:1, the sequences are aligned to compare query sequence 1-15 (RAYFWIAWYKLANSK) to reference sequence 4-18 (RQYFWIAWYKLANSK), thus comparing 14 out of 15 identical amino acids, and providing a calculated 93.3% sequence identity.

[0061] Preferably, the compositions according to the present disclosure are suspensions. A suspension may be defined as a type of a dispersion, i.e., a system having at least one continuous (or coherent) phase and at least one discontinuous (or inner) phase which is dispersed in the continuous phase. In a suspension, the dispersed phase is essentially in the solid state. In one embodiment of the present disclosure, the polypeptide particles are insoluble in the continuous phase, wherein the continuous phase is comprised of the non-aqueous liquid vehicle, and arefeatured in the suspension formulation as the dispersed phase. In a preferred embodiment, the suspension formulations according to the present disclosure are liquid suspensions, at least at physiological temperature, meaning that the continuous phase is a liquid. Typically, the suspensions are also liquid at room temperature.

[0062] The non-aqueous vehicle as used and defined herein may form the continuous phase of a suspension formulation. The non-aqueous vehicle is preferably a liquid at room temperature. As understood herein, the term ‘non-aqueous’ in reference to a vehicle or any formulation component refers a vehicle or formulation component which is essentially free of water. In another embodiment, the non-aqueous vehicle is liquid, and also non-miscible with water. The term ‘a vehicle’ as used herein may refer to a vehicle consisting essentially of only a single component or compound which forms the continuous phase of the suspension formulation, or may refer to a vehicle comprising a combination of two or more components or compounds, which preferably are miscible and form a single continuous phase of the suspension formulation.

[0063] The term ‘particles’ with respect to the polypeptide is understood herein as referring to solid particles comprising the polypeptide that are substantially non-soluble in the non-aqueous vehicle of the suspension formulation, and which are thus are featured as particles dispersed or suspended in the continuous phase formed by the vehicle. The particle as defined herein may comprise the polypeptide in admixture with one or more stabilizing agents, and optionally one or more additional excipients which are combined together in the process of formulating the particle. Preferably, the particle consists of, or consists essentially of, the polypeptide, i.e., without added excipients or other components (e.g., other than trace impurities or degradation products derived from the polypeptide or derived from its synthesis, or trace amounts of buffer).

[0064] Preferably, particles according to the present disclosure have a particle size distribution characterized by characterized by an X90 of less than 25 microns, an X50 of less than 10 microns, 90% of particles having a particle size in the range of 1 to 25 microns, and / or particles having a mean particle diameter of 1 to 10 microns. These parameters are important because they contribute to whether the particles remain stably in suspension, or tend to precipitate, and the surface area of the particles which impacts bioavailability and pharmacokinetics, and whether the suspensions cause eye irritation.

[0065] Particle size analysis methods are established procedures in many laboratories. Various methods are used for this purpose, including laser diffraction (LD), dynamic light scattering(DLS), dynamic image analysis (DIA), and sieve analysis. Some particle size analyzers provide number-based distributions (DIA), others mass-based (SA), and others volume-based particle size distributions (LD). With a suitable model, these distributions can be converted into each other. Preferably, laser diffraction is utilized (e.g., at a wavelength of 632.8 nm and / or measurement range of 0.25-87.5 pm).

[0066] Particle size distribution can generally be represented either in tabular or in graphical form. Typically, particle size is plotted in a graph against percentage (e.g., percent of particles on the Y axis and particle size on the X axis), commonly resulting in a bell-shaped curve. Many statistical parameters can be derived from a particle size distribution, among the most important of which are the percentiles. These indicate in each case the size X below which a certain quantity of the sample lies. Percentiles can be read directly from the particle size distribution curve. Percentiles are denoted by the letter X followed by the % value. Thus, Xio = 83 pm, X50 = 330 pm, and X90 = 1600 pm, mean that 10% of the sample has a particle size smaller than 83 pm, 50% is smaller than 330 pm, and 90% is smaller than 1600 pm. Other notations for these parameters are dl0 / d50 / d90, etc. The X50 value is also called “median” and it divides the particle size distribution into equal amounts of “smaller” and “larger” particles. Usually Xio, X50 and X90 are reported for a particle size distribution. This makes it easy to characterize the middle or central point of the distribution, as well as the upper and lower ends with three values.

[0067] Preferably, the polypeptides and / or polypeptide particles of the present disclosure have a water content (e.g., residual water content, such as after vacuum drying) of less than 12 wt%, based on the total weight of the polypeptide or polypeptide particle. As understood herein, the term ‘water content’ or ‘residual water content’ refers to the amount of water present in a composition (e.g., polypeptide particles), or the amount of water remaining in a composition, such as after processing or manufacturing thereof, which may comprise a step of removal of water. In further embodiments, the suspended polypeptide particles may have a water content of equal to, or less than, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.5%, 0.25%, or 0.15 wt%, based on the total weight of the polypeptide or polypeptide particle.

[0068] Semifluorinated alkanes are linear or branched alkanes some of whose hydrogen atoms have been replaced by fluorine. The semifluorinated alkanes (SFAs) used in the present disclosure are composed of at least one non-fluorinated hydrocarbon segment and at least one perfluorinated hydrocarbon segment and are according to the general formula F(CF2)n(CH2)mH.Another nomenclature which may be used herein refers to the above-mentioned SFAs having two as RFRH, wherein RF designates a perfluorinated hydrocarbon segment, RH designates a non-fluorinated segment. Alternatively, the compounds may be referred to as FnHm, wherein F means a perfluorinated hydrocarbon segment, H means a non-fluorinated segment, and n, and m is the number of carbon atoms of the respective segment. For example, F6H8 is used for 1- perfluorohexyloctane. Moreover, this type of nomenclature is usually used for compounds having linear segments. Such linear SFAs include F(CF2)4(CH2)sH (F4H5), F(CF2)4(CH2)eH (F4H6), F(CF2)6(CH2)6H (F6H6), F(CF2)6(CH2)8H (F6H8), F(CF2)8(CH2)8H (F8H8), and F(CF2)e(CH2)ioH (F6H10) for example. Unless otherwise indicated, it should be assumed that this nomenclature refers to a linear SFA, for example, F3H3 means 1 -perfluoropropylpropane, rather than 2-perfluoropropylpropane, 1 -perfluoroisopropylpropane or 2- perfluoroisopropylpropane.

[0069] In some embodiments, the SFAs of the invention are those of formula F(CF2)n(CH2)mH, in particular, SFAs of the formula F(CF2)4(CH2)5H, F(CF2)4(CH2)6H, F(CF2)6(CH2)6H, F(CF2)6(CH2)8H, andF(CF2)e(CH2)ioH. In particular embodiments the SFA is F(CF2)6(CH2)8H.

[0070] In some embodiments, the composition may further comprise a second SFA which is an SFA of the formula F(CF2)n(CH2)mH, wherein n is an integer from the range of 4 to 8 and m is an integer from the range of 5 to 10 include, in particular, F(CF2)4(CH2)sH, F(CF2)4(CH2)eH, F(CF2)6(CH2)6H, F(CF2)6(CH2)8H, F(CF2)6(CH2)IOH, F(CF2)8(CH2)8H and F(CF2)8(CH2)IOH. In embodiments comprising two SFAs, the SFAs may be present in a weight ratio of at least about 3: 1, for example, at least about 50: 1 or at least about 30: 1, or at least about 10:1.

[0071] Semifluorinated alkanes provide a number of advantages from the pharmaceutical perspective. They are substantially non-toxic and are found to be well-tolerated by various types of human and animal tissue when administered topically or parenterally. In addition, they are chemically inert and are generally compatible with a variety of active and inactive ingredients in pharmaceutical formulations. They are also capable of dissolving a large range of compounds, ranging from small molecule active ingredients to many common pharmaceutically acceptable excipients. Moreover, when used with compounds that are not soluble or poorly soluble (such as some proteins or polypeptides), they tend to form dispersions or suspensions with very useful physical or pharmaceutical properties, such as with little or no tendency to form solid, non- dispersible sediments.

[0072] The use of a semifluorinated alkane vehicle provides a stabilizing effect on the polypeptide compositions of the present disclosure, substantially preventing or inhibiting their aggregation and reducing chemical degradation over a substantial period of time, at room temperature and even at higher temperatures such as 40 °C, without loss of biological activity.

[0073] Dispersions and suspensions of polypeptides in semifluorinated alkanes of the present disclosure also exhibit a remarkable degree of physical stability. The occurrence of flotation or sedimentation takes place slowly, leaving sufficient time for the withdrawal of a dose after gentle shaking or swirling of the container (e.g., a vial) with the dispersion or suspension. The polypeptide particles in the semifluorinated alkane suspension of the present disclosure largely retain their original particle size distribution, and are readily re-dispersible; poorly re-dispersible aggregates do not appear to be formed. Importantly, this provides for a higher level of dosing accuracy in terms of precision and reproducibility.

[0074] In contrast, suspensions or dispersions in other chemically inert vehicles tend to be unstable, leading to formation of dense and poorly re-dispersible aggregates, and making precise dosing challenging, or in some cases, impossible, such as leading to the clogging of fine-gauged needles or dropper channels. Aggregated particles may also present a high risk towards triggering adverse immunogenic reactions.

[0075] In addition, if an ophthalmic suspension settles rapidly after shaking, and if a first dose from a full container is not withdrawn immediately after shaking, a dose that is withdrawn may contain a lower-than-intended number of drug particles (or if the container is held upside down, a larger-than-intended dose will be dispensed). Later doses withdrawn from the same container will also then contain either too high or too low of a drug-dose per volume. Vigorous shaking of polypeptides in attempt to re-disperse poorly re-dispersible aggregates may also further trigger their further aggregation and / or deterioration.

[0076] Liquid SFAs are chemically and physiologically inert, colorless and stable. Their typical densities range from 1.1 to 1.7 g / cm3, and their surface tension may be as low as 19 mN / m. SFAs of the F(CF2)n(CH2)mH type are insoluble in water but also somewhat amphiphilic, with increasing lipophilicity correlating with an increasing size of the non-fluorinated segment.

[0077] SFAs are well-tolerated by the eye, as shown in preclinical testing. In comparison, organic or non-aqueous solvents, perhaps with the exception of oily compounds, are typically very irritating or even highly damaging when administered topically to an eye.

[0078] Moreover, compared to oily carriers or vehicles in ophthalmic compositions for topical use, SFAs exhibit a refractive index in the region of 1.29 to 1.35, which is much better compatible with the aim of a minimally affected vision thus causing little or no blurring.

[0079] Moreover, SFAs exhibit a remarkable wetting and spreading behavior by which they can rapidly and effectively spread over the corneal surface and conjunctiva. This remarkable wetting and spreading behavior permits the SFA to spread away from the administered eye drop rapidly and completely, further permitting the SFA to access the surface of the eye for efficient absorption into the ocular tissues.

[0080] Wetting means the ability of a liquid to establish and maintain contact with a solid surface, resulting from intermolecular interactions when the two are brought together. The balance between adhesive and cohesive forces determines the degree of wetting. The higher the adhesive forces compared to the cohesive forces, the more a drop of liquid will spread across the surface of the solid material. Conversely, very high cohesive forces within the liquid will cause the drop to form a sphere, thus avoiding contact with the surface. Similarly, spreading may also occur at the interface of two liquids which are brought into contact with each other.

[0081] A measure for wetting and spreading is the contact angle 0. The contact angle is the angle at which the liquid-vapor interface meets the solid-liquid or liquid-liquid interface. The tendency of a drop to spread out increases as the contact angle decreases. Thus, the contact angle provides an inverse measure of wettability.

[0082] A low contact angle of less than 90° indicates high wettability and / or spreading, whereas a higher contact angle indicates poor wettability and spreading. Perfect wetting and spreading results in a contact angle of 0°, also reported as no measurable contact angle.

[0083] The enhanced spreading behavior and stable film properties of such ophthalmic compositions comprising SFAs are particularly advantageous for delivery of dissolved or suspended pharmacologic agents. A droplet administered to the surface of the eye may lead to rapid spreading of the SFA mixture compositions over the corneal surface and the formation of a film. A stable film that does not immediately break up provides longer-lasting contact with the ocular surface for drug absorption.

[0084] Another advantage of using ophthalmic compositions comprising SFA is that SFAs are capable of forming very small droplets, for example, of about 10-11 pl volume, when dispensed from a conventional dropper such as a conventional eye dropper. Without wishing to be boundby theory, it is believed that the small droplet size is a result of an interplay of the SFA’s unique properties in terms of their density, viscosity, and surface tension. It is believed that for topical administration into an eye a small drop or volume of administration is highly advantageous as the capability of the lacrimal sac to accept and hold fluid is extremely limited. In fact, it is very common that the administration of a conventional eye drop formulation based on water or oil immediately leads to a discharge of a substantial fraction of the administered medicine as well as some tear fluid. At the same time, there is a risk that some of the administered dose will be taken up systemically via the nasolacrimal duct.

[0085] The present disclosure also provides a means of formulating non-aqueous ophthalmic compositions which are microbiologically stable. Aqueous ophthalmic compositions are prone to bacterial contamination. In comparison, SFAs have bacteriostatic properties and do not support microbial growth. Hence, it is possible to formulate preservative-free ophthalmic compositions which are better tolerable for many patients.

[0086] Optionally, the compositions are administered as a single drop to a tissue of the eye that is topically accessible, preferably the composition of the present disclosure are topically administered to the cornea and / or conjunctiva.

[0087] Optionally the compositions of the present disclosure are highly stable, water-free, and preservative-free.

[0088] In contrast to some other suspensions or dispersions known in prior art, the formulations of the present disclosure require no surfactant, or only small amounts of surfactant, for their physical stabilization. This is a significant advantage as surfactants have a substantial potential for irritation and local toxicity, especially when administered by instillation into the eye. According to one of the preferred embodiments, the compositions of the present disclosure are substantially free of surfactant. In a further embodiment, the total amount of surfactant or surfactants, if more than one surfactant is incorporated, is not more than about 10 wt.-%, in particular not more than about 5 wt.-%, or preferably not more than about 2 wt.-%, respectively. In further preferred embodiments, the amount is not more than about 1 wt.-%, or not more than about 0.5 wt.-%, respectively. In this context, the SFAs as described herein, although they possess some amphiphilic properties due to their chemical structure which includes fluorinated and non-fluorinated alkyl (or alkylene) groups characterized by different degrees of lipophilicity, are not understood as being within the scope of surfactants.

[0089] The surfactants which are absent or only present in small amounts may include non-ionic, cationic, anionic, and zwitterionic surfactants as commonly used as excipients in various types of pharmaceutical compositions, e.g., as wetting agents, emulsifiers, dispersing agents, solubilizers and the like. Examples of surfactants which may be used in the context of the present disclosure include, but are not limited to polysorbates and poloxamers. Poloxamers are triblock copolymers of polyoxyethylene and polyoxypropylene; examples include poloxamer Pl 88, Pluronic F68LF or Lutrol F68, Pluronic L-G2LF and Pluronic L62D. Polysorbates are pegylated sorbitan fatty acid esters; examples which may be useful according to the present disclosure include, but are not limited to polysorbate 20 (polyoxyethylene sorbitan monolaurate), polysorbate 40 (polyoxyethylene sorbitan monopalmitate), polysorbate 60 (polyoxyethylene monostearate) and polysorbate 80 (polyoxyethylene monooleate). In some embodiments, the suspension formulation comprises a surfactant. Other surfactants include polyoxyethylene castor oil derivatives, sorbitan esters, polyoxyl stearates, lecithins, and purified or synthetic phospholipids.

[0090] The compositions of the present disclosure may optionally comprise a non-fluorinated organic liquid, for example in order to modify the properties of the liquid vehicle, such as the viscosity. Such other liquid may be an oil selected from glyceride oils, liquid waxes, and liquid paraffin, or an organic solvent exhibiting a high degree of biocompatibility, or a mixture of more than one liquid excipients.

[0091] Examples of potentially useful oily excipients which may be used in combination with one or more SFA’s include triglyceride oils (e.g., soybean oil, olive oil, sesame oil, cotton seed oil, castor oil, sweet almond oil), mineral oil (e.g., petrolatum and liquid paraffin), medium chain triglycerides (MCT), oily fatty acids, isopropyl myristate, oily fatty alcohols, esters of sorbitol and fatty acids, oily sucrose esters, or any other oily substance which is physiologically tolerated by the eye.

[0092] Examples of potentially useful organic solvents include glycerol, propylene glycol, polyethylene glycol, phenylethyl alcohol, and ethanol. The concentration of the cosolvent should preferably be low relative to that of the SFA or SFA mixture. If an organic solvent such as ethanol is used, it is recommendable to keep it below a level of approximately 5 wt.-%. More preferably, the content of ethanol is from about 0.1 to about 2 wt.-%, or from about 0.1 to 1.4 wt%, and most preferably not more than about 1 wt.-%.

[0093] The composition may of course comprise further pharmaceutical excipients as required or useful. Potentially useful excipients include acids, bases, salts (inorganic or organic), buffers, pH-modifying agents, chelating agents, surfactants, antioxidants, stabilizers, synergists, coloring agents, thickening agents, and - if required in a particular case - preservatives. Any excipients are preferably soluble in or miscible with the semifluorinated alkane(s) used in the non-aqueous liquid vehicle, although insoluble excipients may be incorporated into the polypeptide particles of suspension formulations.

[0094] Generally, however, the invention provides a means of formulating non-aqueous compositions which are microbiologically stable. This is due to the fact that SFAs are not normally prone to microbial contamination. Hence, it is possible to formulate preservative-free compositions to be filled in multi-use containers. Preservative-free compositions are better tolerated by many patients and enable lower costs of final goods.

[0095] The compositions disclosed herein may comprise one or more stabilizing agents, either as part of the suspended particles, or as a component dissolved or dispersed in the non-aqueous liquid vehicle. A ‘stabilizing agent’ as referred to herein may be any excipient, or a combination of two or more excipients, which stabilizes the polypeptide, particle, or suspension. The stabilizing agent may provide a protective effect against mechanical, physical, chemical stress, or a combination thereof during manufacturing processes, or during storage. For example, the stabilizing agent may be useful for preventing instability of the polypeptide during the spraydrying and exposure to temperature extremes, such as elevated temperatures. Examples of stabilizing agents include, but are not limited to, saccharides, polyols, amino acids, amines, surfactants, antioxidants, polymers, salts or combinations thereof.

[0096] In one embodiment, the stabilizing agent is a saccharide or a sugar. The saccharide or sugar may be a monosaccharide, disaccharide, trisaccharide, or optionally an oligosaccharide or a polysaccharide. Examples of saccharides which may function as a stabilizing agent include glucose, fructose, galactose, sucrose, maltose, trehalose, maltose, lactulose, lactose, and cyclodextrins. In one preferred embodiment, the stabilizing agent is selected from trehalose, sucrose, or a combination thereof.

[0097] In another embodiment, the stabilizing agent is a polyol. Examples of polyol include sugar alcohols such as, but not limited to, glycerol, arabitol, erythritol, mannitol, sorbitol, xylitol,maltitol, and lactitol. In yet another embodiment, the stabilizing agent is a saccharide, a polyol, a polysorbate, or a combination thereof.

[0098] In sixth aspect, the present disclosure provides a method of preparing a pharmaceutical composition (e.g., Composition 1, or any of 1.1 et seq.) comprising a polypeptide, or a pharmaceutically acceptable salt thereof, dispersed, or suspended in a non-aqueous liquid vehicle comprising one or more semifluorinated alkanes, wherein the polypeptide is a polypeptide according to SEQ ID NO. 1, or an analog and / or derivative thereof. In preferred embodiment of the sixth aspect, the present disclosure provides a method comprising the steps of:(a) providing the polypeptide, or particles comprising the polypeptide, optionally in admixture with one or more excipients as disclosed herein;(b) optionally drying the polypeptide, or particles comprising the polypeptide, e.g., by vacuum drying and / or heated drying, preferably for at least 12 hours;(c) dispersing, or suspending the polypeptide, or particles comprising the polypeptide from step (a) or step (b) in the non-aqueous liquid vehicle, preferably suspending;(d) optionally adding one or more excipients, e.g., liquid excipients, such as co-solvents, to the dispersion or suspension;(e) optionally adjusting (e.g., reducing or homogenizing) the particle size of the particles, e.g., by sonicating and / or milling the suspension;(f) optionally adding one or more excipients, e.g., liquid excipients, such as co-solvents, to the dispersion or suspension; and(g) optionally packaging the dispersion, or suspension, e.g., in single-use or multiple-use vials, sachets, bottles, or drop dispensers.

[0099] Preferably, the solid particles comprising the polypeptide may be dispersed or suspended in the liquid vehicle comprising the SFA. Alternatively, the particles may be precipitated in situ by adding a - typically organic - solution of the polypeptide (and, optionally, one or more solid excipients) under controlled conditions to the SFA-based vehicle.

[0100] The solid particles may be prepared by lyophilization or spray-drying of a solution of the polypeptide or polypeptide particles. The solution may be aqueous or nonaqueous and may further comprise pharmaceutical excipients as may be useful or required.

[0101] The particle size of the dispersed phase may be also adjusted before or after the particles are combined with the liquid vehicle. In one of the preferred embodiments, particles ofthe polypeptide are provided which already have the appropriately selected particle size. Powders having such selected particle size may be obtained directly from the synthesis of the respective agent by crystal engineering, or after synthesis by conventional grinding or milling methods using standard equipment such as a ball mill, hammer mill, roller mill, colloidal mill, jet mill, or the like. If the particle size is to be reduced after preparation of a suspension, ultrasonication as well as various types of homogenizers may be used, such as colloid mills or high-pressure homogenizers.

[0102] In another embodiment, the method may comprise a step of homogenizing a suspension of the polypeptide in the non-aqueous liquid vehicle formulation. The homogenization may be conducted by any homogenization technique known in the art, e.g., using a high-shear homogenizer, or by ultrasound, which optionally may be conducted under cooling conditions (e.g., under ice-cooling conditions, such as around 0 °C).

[0103] In a preferred embodiment the method includes homogenization carried out using ultrasonication. In a further embodiment, the ultrasonication is performed below ambient temperature, preferably it is performed under cooling, such as under ice-cooling (in an ice bath).

[0104] In another embodiment, the method may comprise an optional step of selecting polypeptide particles with a desired or predetermined particles size, with the particle size being defined by mean particle size diameter. Preferably, said selection of the polypeptide particles with a desired or predetermined particle size may be carried out before suspending the protein particles in the non-aqueous vehicle. The selection of the particles with a desired or predetermined particles size may be carried out by any method known to the skilled person; the selection step may include an additional milling step to generate or increase the number of particles with the desired or predetermined smaller particle size and / or may include a step of sorting out (e.g., by picking or sieving) the particles with the desired or predetermined particle size, to be suspended. The desired or predetermined particle size is defined by the application or medical use of the suspension formulation. Particle size may be determined by laser diffraction.

[0105] The pharmaceutical compositions of the present disclosure form small droplets (drops). In a preferred embodiment, the volume of the composition administered in a single dose per eye is in the range of about 6 to 28 pl, more preferably in the range of about 6 to 24 pl, and most preferably in the range of about 6 to 15 pl, when administered from a suitable drop dispenser. In another preferred embodiment, the volume of the composition administered in asingle dose per eye is in the range of about 8 to 15 pl, preferably in the range of about 9 to 14 pl, most preferably in the range of about 9 to 11 pl, or of about 10 to 12 pl. In a most preferred embodiment, the volume of the composition administered in a single dose per eye is about 10 pl or is about 11 pl.

[0106] As understood herein, the drop dispenser may be a dispenser or applicator means which may be mounted, fixed or connected to the container for holding the pharmaceutical composition. Preferably, the drop dispenser is adapted for dispensing a single dose in the form of a single drop of the pharmaceutical composition according to the first aspect of the invention. More preferably, the drop dispenser is adapted for dispensing a single dose of about 8 pl to about 15 pl volume, preferably of about 9 to 11 pl or of about 10 pl to about 12 pl volume or even more preferably is adapted for dispensing a single dose of about 10 pl, or of about 11 pl volume.

[0107] Such kits as provided in accordance with these embodiments may improve storage and dispensability (i.e., ease and consistency in dispensing) of the pharmaceutical composition.

[0108] Unless otherwise indicated, the term “% (w / v)” as used throughout herein in connection with the present pharmaceutical composition denotes the amount of a component of a composition as a weight percentage in relation to the total volume of the composition (with ‘w’ denoting the weight and ‘v’ denoting volume). For example, 0.05 % (w / v) may be understood as relating to 0.5 mg of a component in 1 mL of the composition, and 0.1 % (w / v) would correspond to 1.0 mg of a component in 1 mL of the composition. Unless otherwise indicated, the term “wt%” or the term “% (w / w)” refers to the amount of a component of a composition as a weight percentage in relation to the total weight of the composition (with ‘w’ denoting weight).

[0109] The term ‘about’ as used herein and in reference or connection to a parameter, for example such as the concentration of polypeptide suspended or dispersed in the composition or the amount of polypeptide featured in a single dose of the composition, includes the precise value as defined, as well as any value falling within the degree of variability usually observed in measuring or determining these parameters using the standard techniques and equipment known in the art and field (e.g., plus or minus 5%).

[0110] In a preferred embodiment, the pharmaceutical composition disclosed herein are substantially free of water. As understood herein, the term ‘substantially free’, or alternatively ‘essentially free’ in reference to a composition constituent refers to the presence of saidconstituent in no more than trace amounts and that if present in trace amounts the constituent provides no technical contribution to the composition.

[0111] The term “administered topically” as used herein comprises all possible methods of administration which allow the present liquid pharmaceutical composition to be brought in contact with a surface of the eye of a subject. Typically, the present pharmaceutical composition may be administered in the form of a single drop or a plurality of drops or droplets to an eye of a subject. The drop may be administered to the surface of the eye, preferably to any surface region or tissue of the eye that is accessible to topical administration or instillation, for example to the cornea or conjunctiva. The drop or droplet of the composition may be instilled directly onto a surface of the eye, such as the corneal surface of the eye, or alternatively into a space, such as a sac or pocket formed by gently pulling down of the lower eyelid of an eye.

[0112] All patents, publications, and other references described herein are hereby incorporated by reference in their entireties.EXAMPLESExample 1: Formulation

[0113] A formulation suitable for topical ophthalmic administration of the polypeptide according to SEQ ID NO. 1 with N-terminal acetylation and C-terminal amidation is prepared. The formulation is a suspension.

[0114] 25.12 mg of the raw synthetic polypeptide (95.8% purity) is weighed into a vial and subjected to vacuum-drying (0.001 mbar, 32 °C) for 22 hours, to provide 23.85 mg of dried polypeptide solid having a density of about 1.2 g / cm3) (5.1 % mass loss upon vacuum-drying). This polypeptide is suspended in 20.85 ml of 1 -perfluorohexyloctane (F6H8). After sealing the vial, the suspension formulation is sonicated for 20 seconds to provide a homogeneous 1 mg / ml suspension, which is free of visible particle agglomerates.

[0115] A 5 mg / ml suspension formulation of is also prepared according to the aforementioned procedure.

[0116] Particle size distribution is measured for both the 1 mg / mL and 5 mg / mL suspensions. The particle size distribution is determined by laser diffraction utilizing a HeNe- laser (wavelength 632.8 nm) at a measurement range of 0.25-87.5 pm (HELOS, Sympatec GmbH, Germany). The results are shown with Xio, Xi6, X50, Xs4, X90 and X99 distributions shown in the Table below:

[0117] These results demonstrate that the prepared polypeptide formulations at both concentrations present as homogeneous suspensions, free of particle aggregates and with particle size distributions suitable for topical administration to the eye. Example 2: Stability Study

[0118] The suspensions of Example 1 are tested for stability. The samples are stored at 25°C / 60% relative humidity (RH) and tested initially, and after 2 weeks, 4 weeks, 2 months, 3 months and 6 months. The tested parameters included (i) appearance and closure integrity (visual inspection), (ii) particle size distribution by laser diffraction (e.g., X50, X90) and (iii) assay measured by UV-HPLC. The particle size distribution is determined by laser diffraction using a HeNe-laser (wavelength 632.8 nm) at a measurement range of 0.25-87.5 pm (HELOS, Sympatec GmbH, Germany). The analysis is performed with on-instrument ultrasonication prior to measurement. UV-HPLC analysis is carried out on a reverse-phase column (Agilent) with UV-detection at 220 nm.

[0119] The X50 and X90 distribution results are shown in Table 1 below:

[0120] The UV-HPLC analysis results are shown in Table 2 below as assay (%) for the peptide:_ Table 2: UV-HPLC Assay for Polypeptide (%)

[0121] Preferably, peptide formulations subjected to accelerated aging conditions will show assay stability of at least 75%, more preferably at least 90%. Without being bound by theory, it is believed small losses in HPLC assay may be attributable to surface effects from the packaging material, rather than the formulation, especially at lower concentrations. These results shown above are consistent with adequate physical and chemical stability under accelerated aging conditions.Example 3: Ischemia Study

[0122] The objective of this study is to investigate whether the suspension formulation of Example 1 can treat or has a neuroprotective effect in the retina from the damage caused by ischemic injury.

[0123] Methodology: Eighteen rats are anesthetized intraperitoneally, and retinal ischemia is induced according to the protocols of Hughes WF, “Quantitation of ischemic damage in the rat retina,” Exp Eye Res 1991; 53: 573-582, and Louzada- Junior P, et al., “Glutamate release in experimental ischaemia of the retina: an approach using microdialysis,” J. Neurochem., 1992; 59: 358 - 363. Intraocular pressure (IOP) is elevated to 155 mmHg for 40 minutes, evoking retinal ischemia. After 40 minutes of ischemia, the cannulating needle is removed and the IOP is normalized. Left eyes are treated with the formulation of Example 1 (10 pl of 1 mg / mL or 5 mg / ml) or vehicle control (F6H8) for 7 days after the ischemic induction. The 10 pl daily doses of 1 mg / mL or 5 mg / ml formulation provide 10 pg per day or 50 pg per day of polypeptide to the eye, respectively. The right eyes served as a non-ischemic control (Group Healthy). The samples are labeled as A, B or C (blinded samples). Six animals are treated with each sample (Groups A, B, and C). After day 7, the animals are euthanized, and the eyes are collected for histological analysis. After unblinding, Sample A is identified as the Example 1 formulation at 5 mg / ml, Sample C is the Example 1 formulation at 1 mg / ml, and Sample B is the vehicle control.

[0124] The thicknesses of the entire retina (between the internal limiting membrane and the retinal pigment epithelial layer), the inner nuclear layer (INL) and the outer nuclear layer (ONL) are measured. The measurements are made at the same distance from the optic disc. The number of cells in the ganglion cell layer (GCL) is calculated (cells per 200 microns). For each eye, at least three measurements at adjacent locations in each hemisphere are made. The representative results for each eye are established as the mean of three or more measurements.

[0125] Results: The results are shown in Table 3 below. Groups A, B, and C indicate results for the retinas of the left eyes for Group A, B, and C animals, respectively (N=6 each), and “Healthy” indicates the results for the retinas of the control right eyes (N=18). Results are expressed as the mean ± standard deviation (* p < 0.05; ** p < 0.01; *** p < 0.001, each in relation to group B):

[0126] The retinas of the left eyes of animals receiving sample A (Ex. 1 ; 5mg / ml) show similar morphology to the healthy group (right eyes). No structural disorder is found. In the group of animals receiving sample B (vehicle control), cellular disorganization between the INL and ONL layers is evident in the left eyes of two animals, and in one animal’s left eye there is observed a massive presence of infiltrating cells and vitreous in a pink color, suggesting inflammation. In addition, a reduction in the number of retinal ganglion cells is observed in most of the retinas of the left eyes of the animals in the B Group. Among animals that received sample C (Ex. 1 ; 1 mg / ml) the total thickness of the left eye retinas is similar between groups. The thickness of the INL differed significantly among the groups. Group B (vehicle control) eyes had the smallest INL thicknesses, statistically significant when compared to Groups A (Group A versus Group B, p = 0.0078), C (Group C versus Group B, p = 0.0205), and Healthy (Healthyversus Group B, p = 0.0221). The thickness of the ONL also differed significantly between groups. Group B eyes had the smallest thickness, statistically significant when compared to Groups A (Group A versus Group B, p = 0.0003) and C (Group C versus Group B, p = 0.0205). Compared to the healthy eyes, the Group B eyes did not show a statistically significant difference, although the reductions are noticeable (Healthy versus Group B, p= 0.0562). Regarding the number of cells in the ganglion layer, a statistically significant reduction was observed in the eyes of Group B when compared with Groups A (Group A versus Group B, p = 0.0028), C (Group C versus Group B, p = 0.0018), and Healthy (Healthy versus group B, p = 0.0082).

[0127] Conclusion: The treatment with the formulation of Example 1 at both concentrations (1 mg / mL and 5 mg / mL) protects retinal cells against ischemic injury, as shown by significant reductions in histological damage. In eyes of Groups A and C, cells from INL and ONL layers are preserved, showing thicknesses and morphology similar to the Healthy control group. Greater preservation of retinal ganglion cells is observed after the ischemic insult using both treatments of Example 1. These findings are not observed in eyes treated with sample B (vehicle control). Therefore, treatment with the formulation of Example 1 (samples A and C) after ischemia induction effectively protected against ischemia-induced retinal damage.Example 4: Ischemia Study with Aqueous Polypeptide Comparison

[0128] The study described in Example 3 is repeated using the same polypeptide (the polypeptide according to SEQ ID NO. 1 with N-terminal acetylation and C-terminal amidation) dissolved in a saline vehicle at a concentration of 4 mg / mL (Ex. 4 formulation), for comparison to the results of Example 3. Test animals are administered either 20 pl per day of the aqueous polypeptide formulation, or vehicle control (saline) for 7 days in the left eyes. Right eyes are used as healthy controls. The daily dose of the aqueous test formulation provides 80 pg per day of polypeptide to the eye. All other parameters and procedures are as described in Example 3.

[0129] For ease of comparison, the results are shown in Table 4 below as “Relative” thicknesses and “Relative” cell numbers. The relative thickness values are calculated by dividing the thickness of the respective retinal layer of the treatment groups by the thickness value of the healthy control group. Likewise, the relative number of retinal ganglion cells is calculated bydividing the number of cells of the treatment group by the number of cells of the healthy control group. The results are shown in Table 4 below:

[0130] The data shows that the relative retinal layer thicknesses and retinal ganglion cell numbers are maintained in both the aqueous formulation treatment group (Table 4A) and the SFA formulation treatment groups (Table 4B), while the relative retinal layer thicknesses and retinal ganglion cell numbers of the vehicle control groups are significant reduced. This demonstrates that the polypeptide provides the same protective effect against ischemia-induced retinal damage using both formulation vehicles, interestingly showing that the polypeptide suspension formulation is comparably effective, as compared to the solution formulation.

[0131] However, it is surprisingly also found that using the SFA-based formulations ofExample 1 provide the same robust protective effects at substantially reduced dosages compared to the aqueous formulation. Specifically, comparable effects are obtained using the 1 mg / mL (10 pg daily) and 5 mg / mL (50 pg daily) SFA-based formulations of Example 1 as are achieved using the 4 mg / mL (80 pg daily) aqueous formulation. The SFA formulations are thus effective at about a 37% lower dose (50 pg) or about an 87% lower dose (10 pg) compared to the aqueous formulation.

[0132] Without wishing to be bound to theory, it is believed that the physico-chemical attributes of the polypeptide suspended in SFA vehicles, in particular in F6H8, may play not only a role in stabilizing the polypeptide, but also in providing improved bioavailability of the polypeptide across the tissues of the eye to the posterior eye segment. The significantly lower administered dose is expected to provide an important reduction in adverse effects, or other unwanted side-effects. This improved efficacy is particularly unexpected since the formulations of Example 1 are suspensions, while the aqueous formulation of Example 4 is a clear, homogenous solution, and it would have been expected that a solution would provide better efficacy and bioavailability.

Claims

Claims1. A pharmaceutical composition comprising a polypeptide, or a pharmaceutically acceptable salt thereof, dispersed, or suspended in a non-aqueous liquid vehicle comprising one or more semifluorinated alkanes, wherein the polypeptide is a polypeptide comprising SEQ ID NO. 1, or an analog and / or derivative thereof.

2. The composition according to claim 1 , wherein the polypeptide is the polypeptide according to SEQ ID NO. 1, without modification (derivatization of any amino acids).

3. The composition according to claim 1, wherein the polypeptide is a derivative of the polypeptide according to SEQ ID NO. 1, wherein the polypeptide comprises one or more modifications of the N-terminal amino group, C-terminal carboxyl group, and / or modifications of the internal amino acid amino and / or carboxyl groups and / or hydroxyl groups (e.g., of the glutamic acid, arginine, lysine, tyrosine, and / or serine residues).

4. The composition according to claim 1, wherein the polypeptide is a polypeptide according to SEQ ID NO. 1, modified with a N-terminal acetyl group and a C-terminal primary amide.

5. The composition according to claim 1, wherein the polypeptide comprises a sequence, or is a polypeptide having a sequence, as provided in any of SEQ ID. NO. 2 through SEQ ID N0.31.

6. The composition according to any one of claims 1-5, wherein the polypeptide is in free form (i.e., not a salt).

7. The composition according to any one of claims 1-5, wherein the polypeptide is in a salt form, e.g., an acetate, chloride, bromide, carbonate, phosphate, palmitate, caproate, or histidate.

8. The composition according to any one of claims 1-7, wherein the non-aqueous liquid vehicle comprises one or more semifluorinated alkanes having the chemical formula F(CF2)n(CH2)mH, wherein n is an integer selected from 4 to 8 and m is an integer selected from 2 to 10, or wherein the non-aqueous liquid vehicle comprises one or more semifluorinated alkanes having the chemical formula F(CF2)n(CH2)mH, wherein n is an integer selected from 4 to 8 and m is an integer selected from 4 to 8.

9. The composition according to any one of claims 1-7, wherein the non-aqueous liquid vehicle comprises one or more semifluorinated alkanes selected from the group consisting of F(CF2)4(CH2)4H, F(CF2)4(CH2)5H, F(CF2)4(CH2)6H, F(CF2)4(CH2)8H, F(CF2)6(CH2)2H, F(CF2)6(CH2)4H, F(CF2)6(CH2)6H, F(CF2)6(CH2)8H, and F(CF2)6(CH2)IOH.

10. The composition according to any one of claims 1-7, wherein the non-aqueous liquid vehicle comprises the semifluorinated alkane F(CF2)g(CH2)8H.

11. The composition according to any one of claims 1-10, wherein the non-aqueous liquid vehicle comprises a linear semifluorinated alkane and a branched semifluorinated alkane of the same formula (i.e., a structural isomer), optionally wherein the branched isomer is present in an amount of 0.1-5% by weight of the linear isomer.

12. The composition according to any one of claims 1-11, wherein the composition is a suspension.

13. The composition according to claim 12, wherein (a) the particles have a particle size characterized by an X90 of less than 50 microns, (b) the particles have a particle size characterized by an X50 of less than 25 microns, (c) 90% of the particles have a particle size characterized by a range of 1 to 50 microns, and / or (d) wherein the particles have a mean particle diameter of 1 to 50 microns.

14. The composition according to claim 12 or 13, wherein the particles have a water content of less than 12%, e.g., as measured by Karl Fischer assay, and / or wherein the particles have a mass loss on vacuum drying of less than 10%, e.g., at 0.001 mbar and 32 °C.

15. The composition according to any one of claims 1-14, wherein the composition comprises the polypeptide at a concentration of about 0.1 to 50 mg / mL, e.g., 0.1 to 25 mg / mL, 0.1 to 20 mg / mL, 0.1 to 15 mg / mL, 0.1 to 10 mg / mL, 0.1 to 8 mg / mL, 0.1 to 6 mg / mL, 0.1 to 5 mg / mL, 0.1 to 4 mg / mL, 0.1 to 3 mg / mL, 0.1 to 2 mg / mL, 0.1 to 1.5 mg / mL, 0.1 to 1.25 mg / mL, 0.5 to 25 mg / mL, 0.5 to 15 mg / mL, 0.5 to 10 mg / mL, 0.5 to 5 mg / mL, 0.5 to 4 mg / mL, 0.5 to 3 mg / mL, 0.5 to 2 mg / mL, 0.5 to 1.5 mg / mL, 0.5 to 1.25 mg / mL, 0.8 to 1.2 mg / mL, 0.9 to 1.1 mg / mL, 1 to 20 mg / mL, 1 to 15 mg / mL, 1 to 10 mg / mL, 1 to 5 mg / ml, 2 to 8 mg / mL, 3 to 7 mg / mL, 4 to 6 mg / mL, 5 to 15 mg / mL, 5 to 10 mg / mL, or about 0.5 mg / ml, or about 1 mg / mL, or about 4 mg / ml, or about 5 mg / mL, or about 10 mg / mL.

16. The composition according to any one of claims 1-15, wherein the composition further comprises one or more excipients selected from organic co-solvents (e.g., ethanol), stabilizing agents, acids, bases, salts (inorganic or organic), buffers, pH-modifying agents, chelating agents, surfactants, antioxidants, stabilizers, synergists, coloring agents, thickening agents, and preservatives.

17. The composition according to any one of claims 1-16, wherein the composition is free or substantially free of water, free or substantially free of surfactants, and / or free or substantially free of preservatives.

18. The composition according to any one of claims 1-17, wherein the composition comprises the polypeptide according to SEQ ID NO. 1, modified with an N-terminal acetyl group and a C-terminal primary amide, suspended in the non-aqueous vehicle comprising the semifluorinated alkane F(CF2)6(CH2)sH, and optionally F(CF2)6(CH)(CH3)(CH2)eH, wherein the composition comprises the polypeptide at a concentration of about 0.1 to 5 mg / mL (e.g., about 1 mg / mL or about 5 mg / mL), and the semifluorinated alkane(s) in an amount of 95-99% by weight of the liquid vehicle.

19. The composition according to any one of claims 1-18, for use as a medicament, e.g., for use as a topical ophthalmic medicament, for example, for use in a method of treating or preventing a posterior eye disorder (e.g., diabetic retinopathy), or for use in a method of treating or preventing a retinal eye disorder.

20. The composition according to any one of claims 1-18, for use in a method of treating or preventing a posterior eye disorder (e.g., diabetic retinopathy) or a retinal eye disorder.

21. The composition according to any one of claims 1-18, for use in a method of inducing nitric oxide synthase or increasing nitric oxide production in the tissues of the posterior eye (e.g., the retina, macula, and / or optic nerve).

22. A kit comprising a pharmaceutical composition according to any one of claims 1-18, and a container for holding the pharmaceutical composition and a drop dispenser for administering the composition, and optionally further comprising instructions for use of said composition as a medicament or for use in a method of treating or preventing a posterior eye disorder (e.g., diabetic retinopathy) or for use in a method of inducing nitric oxide synthase or increasing nitric oxide production in the tissues of the posterior eye.

23. A method of preparing a pharmaceutical composition according to any one of claims 1-18, comprising the steps of:(a) providing the polypeptide, or particles comprising the polypeptide, optionally in admixture with one or more excipients as disclosed herein;(b) optionally drying the polypeptide, or particles comprising the polypeptide, e.g., by vacuum drying and / or heated drying, preferably for at least 12 hours; (c) dispersing, or suspending the polypeptide, or particles comprising the polypeptide from step (a) or step (b) in the non-aqueous liquid vehicle, preferably suspending;(d) optionally adding one or more excipients, e.g., liquid excipients, such as co-solvents, to the dispersion or suspension;(e) optionally adjusting (e.g., reducing or homogenizing) the particle size of the particles, e.g., by sonicating and / or milling the suspension;(f) optionally adding one or more excipients, e.g., liquid excipients, such as co-solvents, to the dispersion or suspension; and(g) optionally packaging the dispersion, or suspension, e.g., in single-use or multiple-use vials, sachets, bottles, or drop dispensers.

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