Peptide compositions and methods of use

Abstract

Provided herein are compositions comprising peptides, pharmaceutical preparations thereof, and methods of using the same to prevent photoreceptor death and protect retinal cells (including but not limited to photoreceptors and retinal pigment epithelium) from Fas- or TRAIL-mediated apoptosis.

Description

[0001] This application is a divisional application of a patent application filed on April 29, 2016, with application number 201680038258.8 and invention name “Peptide Compositions and Methods of Use”.

[0002] All patents, patent applications, and publications, as well as other literature references, cited herein are hereby incorporated by reference in their entirety. The disclosures of these publications are hereby incorporated by reference in their entireties into this application in order to more fully describe the state of the art as known to those skilled in the art as of the date of the invention described and claimed herein.

[0003] Federally sponsored research or development

[0004] This invention was made with government support under Grant No. R44EY022512 awarded by the National Institutes of Health (NIH). The government has certain rights in this invention. Field of the Invention

[0005] Described are compositions for protecting cells, particularly retinal cells (including but not limited to photoreceptors, retinal pigment epithelium (RPE), and retinal ganglion cells, which receive visual information from photoreceptors) from cell death mediated by extrinsic pathways (such as Fas-mediated apoptosis, TRAIL-mediated apoptosis, TNF-mediated necrosis, and pyroptosis), and methods of using the compositions. Background Art

[0006] Several major causes of vision loss, such as retinal detachment, glaucoma, and macular degeneration, have an important component of apoptotic signaling, which in turn leads to the programmed cell death of certain very important cell types in the retina. Three of these cell types are the retinal pigment epithelium (the loss of which can be seen in retinal bleaching, retinitis pigmentosa, and age-related dry macular degeneration), the retinal ganglion cells (the loss of which can be seen in glaucoma), and the photoreceptor cells themselves (the primary visual signaling cells, and whose loss is the ultimate cause of vision loss in retinal diseases).

[0007] Retinal detachment (RD) is defined as the separation of the neurosensory retina from the underlying RPE, which results in the apoptotic death of photoreceptor cells (Cook et al. 1995; 36(6):990-996; Hisatomi et al. Curr Eye Res. 2002; 24(3):161-172; Zacks et al. Invest Ophthalmol Vis Sci. 2003; 44(3):1262-1267. Yang et al. Invest Ophthalmol Vis Sci. 2004; 45(2):648-654; herein incorporated by reference in their entireties). Rodent and feline models of RD have demonstrated that pro-apoptotic pathways are activated almost immediately after separation of the retina from the RPE (Cook et al. 1995; 36(6):990-996; Hisatomi et al. Curr Eye Res. 2002; 24(3):161-172; Zacks et al. Invest Ophthalmol Vis Sci. 2003; 44(3):1262-1267. Yang et al. Invest Ophthalmol Vis Sci. 2004; 45(2):648-654; herein incorporated by reference in their entireties). Histological markers of apoptosis, such as terminal deoxynucleotidyl transferase nick end labeling (TUNEL) staining, peak approximately 3 days after RD, with persistent apoptotic activity and progressive cell death during detachment. This has also been demonstrated in human retinal detachment (Arroyo et al. Am J Ophthalmol. 2005 Apr; 139(4):605-10). However, clinical experience with repairing retinal detachment shows that there is a window of opportunity for repair while retaining a certain degree of visual acuity, but as the time between detachment and repair increases, visual acuity decreases significantly (Burton. Trans Am Ophthalmol Soc. 1982; 80: 475-497; Ross et al. Ophthalmology. 1998; 105(11): 2149-2153; Hassan et al. Ophthalmology. 2002; 109(1): 146-152; incorporated herein by reference in their entirety). The rapid activation rate of pro-apoptotic pathways and the slower rate of vision loss suggest that intrinsic neuroprotective factors may be activated within the neural retina and may serve to counteract the effects of pro-apoptotic pathways activated by retinal RPE detachment.

[0008] Age-related macular degeneration (AMD) is the main cause of permanent vision loss in the United States (Bourne et al.BrJ Ophthalmol.2014; 98: 629-638; Klein et al.Arch Ophthalmol.2011; 129: 75-80; Cruciani et al.Clin Ter.2011; 162: e35-42). The death of the outer retina (defined herein as a complex of retinal pigment epithelium (RPE) and photoreceptor (PR) cells) is the root cause of AMD vision loss and limits the effectiveness of current treatments (Murakami et al.Prog Retin Eye Res.2013; 37: 114-140; Huckfeldt and Vavvas.Int Ophthalmol Clin.2013; 53: 105-117). The interruption of PR-RPE homeostasis leads to PR death. Compared with healthy controls, Fas is significantly expressed in the eyes of people's late stage AMD that is defined as wet or atrophic, and is most concentrated in active angiogenesis and atrophic lesions around (Dunaief et al.Arch Ophthalmol.2002; 120:1435-1442). RPE is sensitive to the apoptosis of the Fas-mediation under the stress conditions (such as inflammation or oxidative stress) that occur during AMD development, and compared with the healthy counterpart of age matching, in AMD patients, identify higher concentration of soluble Fas ligand (Jiang et al.Invest Ophthalmol Vis Sci.2008; 37:114-140). Similarly, the oxidative stress that occurs during AMD progression causes the expression of Fas in RPE to increase (Lin et al.Invest Ophthalmol Vis Sci. 2011; 52: 6308-6314), and the death of the RPE that occurs under oxidative stress conditions depends on Fas signal conduction (Wang et al.Apoptosis. 2012; 17: 1144-1155). In addition, Fas is directly related to the RPE cell death induced by Alu RNA accumulation, and Alu RNA accumulation is another generally acknowledged factor (Kim et al.Proc Natl Acad Sci USA. 2014 of AMD pathology; 111: 16082-16087). It has been shown that the TRAIL-R1 receptor (DR4) operated in part by the same pathway is a genetic risk factor (DR4) of TRAIL-R1 receptor (DR4), and the TRAIL-R1 receptor (DR4) operated in part by the same pathway is a genetic risk factor for age-related macular degeneration.(Miyake et al. Invest Ophthalmol VisSci 56, 5353 (2015)).

[0009] Fas has also been implicated in retinal ganglion cell death associated with glaucoma (Gregory et al. PLoS One. 2011; 6(3): e17659). Furthermore, intraocular pressure (IOP) is a major risk factor for glaucoma progression, and animal models of IOP demonstrate increased expression of Fas and FasL (Ju et al. Brain Res. 2006; 1122(1): 209-221) and retinal ganglion cell death by apoptosis (Ji et al. Vision Res. 2005; 45(2): 169-179). Although controlling intraocular pressure is a major principle of clinical glaucoma treatment, a significant number of patients continue to experience disease progression even after adequate IOP control, and additional work has reinforced the idea that other contributing factors to glaucoma may need attention (Kamat et al. Semin Ophthalmol. 2016; 31(1-2): 147-154).

[0010] Apoptosis (programmed cell death) plays a central role in the development and homeostasis of all multicellular organisms. Alterations in apoptotic pathways have been implicated in many types of human pathologies, including developmental disorders, cancer, autoimmune diseases, as well as neurodegenerative disorders and retinal degeneration. It is a tightly regulated pathway that controls the death process of individual cells and can be initiated either extrinsically or intrinsically. The latter is an intracellular mechanism triggered by mitochondria, while the former involves the interaction of "death receptors" with their corresponding ligands on the cell membrane. Therefore, programmed cell death pathways have become attractive targets for the development of therapeutic agents. In particular, attention has focused on anticancer therapies using pro-apoptotic agents, as killing cells is conceptually easier than maintaining them. However, there are many diseases in which inappropriate activation of apoptotic pathways leads to tissue degeneration, and treatments must be designed to block the activation of intrinsic or extrinsic apoptotic pathways in this specific disease pathology. The Fas receptor is the most common of the death receptors involved in apoptosis in retinal degenerative diseases (Chinsky et al. Curr Opin Ophthalmol. 2014 25(3); 228-233). Fas is a typical cytokine cell surface receptor that is activated by trimerization when it binds to its trimeric cognate ligand, FasL. Stress following RD modulates Fas receptors on retinal cells (e.g., photoreceptors). Invading immune cells induced by stress express the transmembrane protein Fas ligand (FasL) on their surface. FasL binds to the Fas receptor on retinal cells, leading to rapid activation of the extrinsic cell death pathway, and the signal is transmitted through a cascade reaction. Initially, the "initiator" caspase-8 is cleaved into an active form, thereby activating caspase-3, the downstream "executioner" of the apoptotic cell death pathway. However, in the eyes of mice infected with mouse cytomegalovirus, Fas and the related death receptors TNFR1 and TRAIL have been shown to be activated, and this activity can lead to apoptosis, necrosis, and pyroptosis in ocular cells (Chien and Dix J Virol 86, 10961 (2012)).

[0011] It has been shown that photoreceptor cells in culture are very sensitive to apoptosis induced by FasL, indicating that FasL-induced apoptosis is a major contributing factor to vision loss in retinal diseases (Burton. Trans Am Ophthalmol Soc. 1982; 80: 475-497; Ross et al. Ophthalmology. 1998; 105(11): 2149-2153; Hassan et al. Ophthalmology. 2002; 109(1): 146-152.). In addition, the Fas receptor, Met-12, H-12, and FasL-binding domains of the oncoprotein Met are also involved.60 HIYLGAVNYIY 71 (SEQ ID NO: 2) The small peptide inhibitor Zou et al. Nature Medicine 13, 1078 (2007)) has been shown to be photoreceptor protective in cell culture experiments and in the context of isolated retina and retinal pigment epithelium and other ocular conditions or diseases (Besirli et al., Invest Ophthalmol Vis Sci., 51(4):2177 - 84 (2010); U.S. Pat. No. 8,343,931; incorporated herein by reference in its entirety). In addition, c - Met, which may use the same binding domain homologous to Met - 12, FasL, TNAα and TRAIL, has been shown to block TRAIL - induced apoptosis in various tumors (Du et al. PLoS One 9, e95490 (2014)).

[0012] The Met - 12 peptide itself has biopharmaceutical properties, which are mainly determined by its extremely poor water solubility. Experiments have clearly shown that Met - 12 must be administered as a solution in vitro and in vivo to display optimal activity, and it has been demonstrated that it is very difficult to produce such a solution in a large amount of aqueous medium, especially under conditions acceptable for intravitreal injection. The administration of a suspension or gel of Met - 12 results in a great loss of potency. For example, even a visibly clear solution of 10 mg / mL of Met - 12 in 20 mM citrate buffer (pH 2.8) shows a considerable loss of material upon filtration and results in a loss of activity of at least five - fold when used in the in vitro and in vivo assays described below. Despite a large amount of development work, the only solution formulations of Met - 12 that have been found involve some solution injectables with extremely low pH (<pH 2.8) or pure DMSO injectables, all of which are not optimal for intravitreal injection.

[0013] Thus, there is still a need for a peptide composition that meets the following requirements to help maintain vision: it protects retinal cells (including but not limited to photoreceptors, retinal ganglion cells, and retinal pigment epithelium) from cell death by exogenous pathways (including Fas - and TRAIL - mediated apoptosis); it is easy to formulate in a solution or suspension that can be delivered to the eye in a manner that allows sufficient contact without using excipients that may cause ocular (or other) toxicity; and it is easy to use. SUMMARY OF THE INVENTION

[0014] This article provides a pharmaceutical product of a bioactive aqueous formulation of a photoreceptor - protecting peptide, its pharmaceutical product, and methods for preventing photoreceptor death and treatment methods using the same.

[0015]

[0016] Compound 1. His-His-Ile-Tyr-Leu-Gly-Ala-Val-Asn-Tyr-Ile-Tyr-amide (SEQ ID NO: 1).

[0017] Some embodiments relate to a C-terminal amide peptide (Compound 1 (above)) or a pharmaceutically acceptable salt thereof. Certain other embodiments relate to a polyacetate salt of Compound 1. Certain other embodiments relate to a triacetate salt of Compound 1. The compound can be used in a pharmaceutical formulation for preventing Fas- or TRAIL-mediated apoptosis in the photoreceptors of the eye. The compound can be used in a pharmaceutical formulation for preventing Fas-mediated apoptosis in the retinal pigment epithelium of the eye. The compound can be used in a pharmaceutical formulation for treating retinal detachment. The compound can be used in a pharmaceutical formulation for treating retinal ganglion cell diseases, such as glaucoma.In some other embodiments, the compounds can be used in pharmaceutical formulations for treating ocular diseases or conditions including: maculopathy / retinal degeneration, such as macular degeneration, including age-related macular degeneration (AMD), such as non-exudative AMD and exudative AMD; choroidal neovascularization; retinopathy, including diabetic retinopathy, acute and chronic macular neuroretinopathy, central serous chorioretinopathy; and macular edema, including cystoid macular edema, diabetic macular edema; uveitis / retinitis / choroiditis, such as acute multifocal squamous pigment epitheliopathy, Behcet's disease, birdshot retinopathy, and retinal edema. retinochoroidopathy), infectious diseases (syphilis, Lyme disease, tuberculosis, toxoplasmosis), uveitis such as intermediate uveitis (pars planitis) and anterior uveitis, multifocal choroiditis, multiple transient white dot syndrome (MEWDS), ocular sarcoidosis, posterior scleritis, creeping choroiditis, subretinal fibrosis, uveitis syndrome, and Vogt-Koyanagi-Harada syndrome; vascular diseases / exudative diseases such as retinal artery occlusive disease, central retinal vein occlusion, disseminated intravascular coagulopathy, branch retinal vein occlusion, hypertensive fundus changes, ocular ischemic syndrome, retinal artery microaneurysms, outer exudative retinopathy (Coats' disease), and ocular ischemic retinopathy. disease), parafoveal telangiectasia, hemiretinal vein occlusion, optic disc phlebitis, central retinal artery occlusion, branch retinal artery occlusion, carotid artery disease (CAD), frosty dendritic vasculitis, sickle cell retinopathy and other hemoglobinopathies, angioid streaks, familial exudative vitreoretinopathy, Eales' disease, trauma / surgical conditions: sympathetic ophthalmia, uveitic retinopathy, retinal detachment, trauma, laser, PDT, photocoagulation, hypoperfusion during surgery, radiation retinopathy, bone marrow transplant retinopathy; proliferative diseases such as proliferative vitreoretinopathy and epiretinal membranes, proliferative diabetic retinopathy.Infectious diseases: ocular histoplasmosis, ocular toxocariasis, ocular histoplasmosis syndrome (OHS), endophthalmitis, toxoplasmosis, retinopathy associated with HIV infection, choroidopathy associated with HIV infection, uveopathy associated with HIV infection, viral retinitis, acute retinal necrosis, progressive outer retinal necrosis, fungal retinal disease, ocular syphilis, ocular tuberculosis, diffuse unilateral subacute neuroretinitis, and myiasis; genetic diseases such as retinitis pigmentosa, systemic disorders associated with retinal dystrophy, congenital stationary night blindness, cone dystrophy, Stargardt's disease, fundus macularis, congenital macular degeneration (Best's disease), pattern dystrophy of the retinal pigment epithelium, X-linked retinoschisis, Sorsby's fundus dystrophy dystrophy), benign concentric maculopathy, Bietti lens dystrophy, pseudoxanthoma elasticum. Retinal tears / holes: retinal detachment, macular hole, giant retinal hole, such as retinopathy associated with tumors, congenital hypertrophy of the RPE, posterior uveal melanoma, choroidal hemangioma, choroidal osteoma, choroidal metastatic carcinoma, combined hamartoma of the retina and retinal pigment epithelium, retinoblastoma, fundus vascular proliferative tumor, retinal astrocytoma, intraocular lymphoid tumor; and other diseases and conditions such as punctate inner choroidopathy, acute posterior multifocal squamous pigment epitheliopathy, myopic retinal degeneration, acute retinal pigment epitheliitis, corneal dystrophy or developmental abnormalities, etc.

[0018] Further embodiments relate to compositions comprising compound 1 or a pharmaceutically acceptable salt thereof (e.g., polyacetate and triacetate) and a pharmaceutical carrier formulated for ocular delivery. The composition can be formulated for intraocular, intravitreal, or periocular administration. Compound 1 or a pharmaceutically acceptable salt thereof protects detached retinal photoreceptor cells in the composition. Compound 1 or a pharmaceutically acceptable salt thereof prevents cell death of extrinsic pathways in the composition, including apoptosis in the retinal pigment epithelium of the eye. Compound 1 or a pharmaceutically acceptable salt thereof prevents diseases of retinal ganglion cells, such as glaucoma, in the composition. The composition is sterile, non-pyrogenic, and non-toxic to the eye. The composition may further comprise at least one nonionic surfactant. The at least one nonionic surfactant may be polysorbate 80, polysorbate 20, poloxamer 407, and tyloxapol, but is not limited to these examples. The at least one nonionic surfactant may comprise approximately 0.01%-20% w / w of the composition; or approximately 0.05%-10% w / w of the composition; or approximately 0.1%-3% w / w of the composition. Alternatively, a mixture of nonionic surfactants may be used, wherein at least two of the aforementioned or other nonionic surfactants are used together in a ratio that optimizes the desired pharmacokinetics of the formulation, wherein the total amount of surfactant falls within the aforementioned limits. The composition may further comprise an organic cosolvent, such as propylene glycol or dimethyl sulfoxide. The organic cosolvent may comprise approximately 1%-50% w / w of the composition; or approximately 1%-20% w / w of the composition; or approximately 1%-5% w / w of the composition. An isotonicity agent, such as trehalose, mannitol, or sorbitol, or a soluble inorganic salt, such as NaCl, may also be added to bring the osmotic pressure of the solution into the range of 250-400 mOsm / L. The composition may have a pH in the range of 2.5-6.0 and may be buffered by methods known to those skilled in the art.

[0019] Another embodiment is directed to a method of treating an ocular condition, disease, or condition or disease affecting ocular health, comprising administering to a subject suffering from the ocular condition, disease, or condition or disease affecting ocular health a composition comprising Compound 1 or a pharmaceutically acceptable salt thereof and a pharmaceutical carrier formulated for optical delivery. The ocular condition, disease, or condition or disease affecting the health of the eye may be retinal detachment, macular degeneration, age-related macular degeneration, non-exudative age-related macular degeneration, exudative age-related macular degeneration, choroidal neovascularization, retinopathy, diabetic retinopathy, acute and chronic macular neuroretinopathy, central serous chorioretinopathy, macular edema, cystoid macular edema, diabetic macular edema, uveitis / retinitis / choroiditis, multifocal squamous pigment epitheliopathy, Behçet's disease, retinochoroidopathy, infectious (syphilis, Lyme disease, tuberculosis, toxoplasmosis), uveitis, intermediate uveitis (pars planitis), anterior uveitis, multifocal choroiditis, multiple transient white dot syndrome (MEWDS), ocular sarcoidosis, posterior scleritis, creeping choroiditis, subretinal fibrosis, uveitis syndrome, Vogt-Koyanagi-Harada syndrome;Retinal artery occlusive disease, central retinal vein occlusion, disseminated intravascular coagulopathy, branch retinal vein occlusion, hypertensive fundus changes, ocular ischemic syndrome, retinal artery microaneurysms, outer exudative retinopathy, parafoveal telangiectasia, hemiretinal vein occlusion, optic disc phlebitis, central retinal artery occlusion, branch retinal artery occlusion, carotid artery disease (CAD), frosty dendritic vasculitis, sickle cell retinopathy and other hemoglobinopathies, angioid streaks, familial exudative vitreoretinopathy, Eales' disease, sympathetic ophthalmia, uveitic retinopathy, retinal detachment, trauma during surgery, laser, PDT, photocoagulation, hypoperfusion, radiation retinopathy, bone marrow transplant retinopathy, proliferative vitreoretinopathy and epiretinal membrane, proliferative diabetes mellitus ocular histoplasmosis, ocular toxocariasis, ocular histoplasmosis syndrome (OHS), endophthalmitis, toxoplasmosis, retinopathy associated with HIV infection, choroidopathy associated with HIV infection, uveopathy associated with HIV infection, viral retinitis, acute retinal necrosis, progressive outer retinal necrosis, fungal retinal disease, ocular syphilis, ocular tuberculosis, diffuse unilateral subacute neuroretinitis, myiasis, retinitis pigmentosa, systemic disorders associated with retinal dystrophy, congenital stationary night blindness, cone dystrophy, Stargardt's disease, fundus xanthophyta, congenital macular degeneration, retinal pigment epithelial dystrophy, X-linked retinoschisis, Sorsby's dystrophy fundus dystrophy), benign concentric maculopathy, Bietti lens dystrophy, pseudoxanthoma elasticum, retinal detachment, macular hole, giant retinal hole, tumor-related retinopathy, congenital hypertrophy of the RPE, posterior uveal melanoma, choroidal hemangioma, choroidal osteoma, choroidal metastatic carcinoma, combined hamartoma of the retina and retinal pigment epithelium, retinoblastoma, fundus vascular proliferative tumor, retinal astrocytoma, intraocular lymphoma, punctate inner choroidopathy, acute posterior multifocal squamous pigment epitheliopathy, myopic retinal degeneration, abnormal retinal pigment epithelial cell homeostasis, acute retinal pigment epitheliitis, glaucoma, corneal dystrophy or developmental abnormalities, etc. The composition can be administered in an amount sufficient to attenuate cell death in a subject. The composition can be administered in an amount sufficient to enhance photoreceptor survival in the subject. The composition can be administered in an amount sufficient to protect retinal pigment epithelial cells in the subject. The composition can be administered in an amount sufficient to protect retinal ganglion cells in the subject.

[0020] Another embodiment relates to a method for preventing photoreceptor, RPE or retinal ganglion cell death, comprising administering to a subject a composition comprising compound 1 or a pharmaceutically acceptable salt thereof and a sterile non-pyrogenic pharmaceutical carrier. Photoreceptor, RPE or retinal ganglion cell death is a photoreceptor or RPE cell apoptosis mediated by Fas. The subject may have a risk of photoreceptor, RPE or retinal ganglion cell death. The composition can be administered to the subject intraocularly, intravitreally or periocularly.

[0021] Another embodiment relates to a method for increasing photoreceptor, RPE or retinal ganglion cell survival, the method comprising administering a photoreceptor, RPE or retinal ganglion protective composition comprising compound 1 or its pharmaceutically acceptable salt. The increase in photoreceptor, RPE or retinal ganglion cell survival comprises suppressing photoreceptor, RPE or retinal ganglion cell apoptosis. Photoreceptor, RPE or retinal ganglion cell death comprises apoptosis of the photoreceptor, RPE or retinal ganglion cell mediated by Fas. Photoreceptor, RPE or retinal ganglion cell death comprises apoptosis of the photoreceptor, RPE or retinal ganglion cell mediated by TRAIL. Photoreceptor, RPE or retinal ganglion cell death comprises necrosis of the photoreceptor, RPE or retinal ganglion cell mediated by TNFR. Photoreceptor, RPE or retinal ganglion cell death comprises pyroptosis of the photoreceptor, RPE or retinal ganglion cell mediated by exogenous pathway. The composition can be administered by intravenous, subcutaneous or intramuscular injection, or orally or topically, ie, intraocularly, intravitreally, topically, suprachoroidally, subconjunctivally, subretinally or periocularly. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Figure 1 shows a graph depicting the blocking of Fas-induced caspase-8 activation by Met-12 and Compound 1 trihydrochloride in 661W cells. 661W cells were pretreated with varying amounts of Met-12 or Compound 1 dissolved in DMSO, both at 20 mg / mL, for 1 hour. FasL (500 ng / mL) was then added, and caspase-8 activity was measured 48 hours after treatment with FasL.

[0023] Figure 2Shown is a graph depicting the blocking of Fas-induced caspase 8 activation by Met-12 and compound 1 trihydrochloride in 661W cells. 661W cells were pretreated for 1 hour with varying amounts of Met-12 in DMSO (circles), compound 1 in DMSO (20 mg / mL) (diamonds), and compound 1 in a 2% polysorbate (PS) 20, 2% propylene glycol (PG) formulation at pH 4 (triangles), all at 10 mg / mL. Cells were then treated with FasL (500 ng / mL), and caspase 8 activity was measured 48 hours after treatment with FasL.

[0024] Figure 3 Shown is a graph depicting the blocking of Fas-induced caspase 8 activation by compound 1 trihydrochloride in 661W cells. 661W cells were pretreated for 1 hour with varying amounts of compound 1 in DMSO (20 mg / mL) (circles), in a formulation containing 3% polysorbate 20, 3% propylene glycol at pH 4 (triangles), and in a formulation containing 1% polysorbate 20, 3% propylene glycol at pH 4 (diamonds), all at a concentration of 10 mg / mL. Cells were then treated with FasL (500 ng / mL), and caspase 8 activity was measured 48 hours after treatment with FasL.

[0025] Figure 4 A graph depicting the blocking of Fas-induced caspase 8 activation by compound 1 trihydrochloride in 661W cells is shown. 661W cells were pretreated with varying amounts of compound 1 trihydrochloride in DMSO (20 mg / mL) (circles) or in a formulation of 0.4% polysorbate-20, 4.5% mannitol, 10 mM acetate, pH 4 (triangles), all at a concentration of 2 mg / mL. Cells were then treated with FasL (500 ng / mL), and caspase 8 activity was measured 48 hours after treatment with FasL.

[0026] Figure 5a Depicted are logarithmic plots of rabbit retinal concentrations of Compound 1 delivered intravitreally in three poloxamer formulations.

[0027] Figure 5b Logarithmic plots of rabbit retinal concentrations of intravitreally delivered Compound 1 are depicted to compare a poloxamer 407-based formulation to a polysorbate-20-based formulation over time.

[0028] Figure 6a Depicted are linear graphs of rabbit vitreous humor (VH) concentrations over time for Compound 1 delivered intravitreally in three poloxamer formulations at varying concentrations of surfactant (0.4% or 0.1%) and varying concentrations of Compound 1 (2 mg / mL vs. 0.5 mg / mL).

[0029] Figure 6b Depicted are linear graphs of rabbit vitreous humor (VH) concentrations over time for different choices of surfactant (0.4% poloxamer 407 versus 0.4% polysorbate 20) and different amounts of Compound 1 (2 mg / mL versus 1 mg / mL).

[0030] Figure 7 Shown are the total amount of Compound 1 triacetate in the vitreous humor (dark) and the concentration in the retina (light) of Brown Norway rats 24 and 72 hours after a nominal injection of 300 ng of Compound 1 triacetate (5 μL, 0.06 mg / mL) in 4.5% mannitol, 10 mM acetic acid, 0.4% poloxamer (PX) 407, pH 4.5.

[0031] Figure 8 Shown are bar graphs depicting the number of apoptotic cells in detached retinas of rats treated in vivo with Met-12 trihydrochloride (light bars) (5 μg in 5 μL DMSO), and with Compound 1 trihydrochloride (dark) (0.5, 1.0, 5, and 10 μg in 5 μL DMSO) or DMSO vehicle (light). The LHS bars are undetached control retinas that were not injected.

[0032] Figure 9 Shown is a bar graph depicting the number of apoptotic cells in detached retinas of rats treated in vivo with Compound 1 trihydrochloride (1.0 and 5 μg in 5 μL F1 or F2) versus the same substances in DMSO (5 μg in 5 μL) or DMSO vehicle (grey) for 72 hours. The LHS bar graph is a non-detached control retina that was not injected. F1 (5 / 1 μg) is 1.0 / 0.2 mg / mL Compound 1 trihydrochloride in 3% PG / 3% PS-20, pH 4.0 (black), and F2 (5 / 1 μg) is 1.0 / 0.2 mg / mL Compound 1 trihydrochloride in 2% PG / 2% PX-407, pH 4.0 (vertical bars).

[0033] Figure 10 Shown is a bar graph depicting the percentage of apoptotic cells in detached retinas from rats treated in vivo 72 hours after treatment. Figure 1 is the vehicle control in the detached retina. Figure 2 0.2 mg / mL DMSO solution containing 1 μg of compound 1 triacetate. Figure 3 0.2 mg / mL DMSO solution containing 1 μg of compound 1 trihydrochloride. Figure 4 are uninjected, non-detached control retinas. DETAILED DESCRIPTION

[0034] All patents, patent applications, and publications, as well as other literature references, cited herein are hereby incorporated by reference in their entirety. The disclosures of these publications are hereby incorporated by reference in their entireties into this application in order to more fully describe the prior art as known to those skilled in the art as of the date of the invention described and claimed herein.

[0035] Bioactive peptide compositions, pharmaceutical formulations of the bioactive peptide compositions, and methods of using the peptide compositions are described.

[0036] The term "therapeutically effective amount" refers to an amount of a drug or agent (e.g., Compound 1) that is effective in promoting a desired therapeutic effect in a specific class of subjects (e.g., infants, children, adolescents, adults). As used herein, the term "subtherapeutic" refers to an amount of a drug or agent that is insufficient to achieve a desired and / or expected therapeutic outcome / result after administration to an average and / or typical subject (e.g., of average size, not taking contraindications, having a dose response similar to that of the majority of the population, etc.). Doses recommended by the U.S. Food and Drug Administration (FDA) represent therapeutic doses.

[0037] As used herein, the term "drug" or "pharmaceutical agent" refers to a compound, peptide, macromolecule, or other entity (e.g., in the context of a pharmaceutical composition) that is administered to a subject to elicit a desired biological response. An agent can be a "drug" or any other substance (e.g., a peptide, polypeptide) that has biological activity in humans or other mammals, locally and / or systemically. Examples of drugs are disclosed in the Merck Index and the Physicians Desk Reference, the entire disclosures of which are incorporated herein by reference for all purposes.

[0038] As used herein, the term "pharmaceutical preparation" refers to a combination of at least one medicament (e.g., Compound 1) and one or more additional ingredients that contribute to making the medicament suitable for achieving the desired effect after being administered to a subject. The pharmaceutical preparation may include one or more additives, such as pharmaceutically acceptable excipients, carriers, penetration enhancers, coatings, stabilizers, buffers, acids, bases, or other substances physically associated with the medicament to enhance the administration, release (e.g., time of release), delivery rate, bioavailability, efficacy, etc. of the dosage form. The preparation may be, for example, a liquid, suspension, solid, nanoparticle, emulsion, micelle, ointment, gel, emulsion, coating, etc. The pharmaceutical preparation may include a single medicament (e.g., Compound 1) or multiple medicaments. The pharmaceutical composition may contain a single pharmaceutical preparation or multiple pharmaceutical preparations. In some embodiments, a medicament (e.g., Compound 1) is formulated for a specific mode of administration (e.g., ocular administration (e.g., intravitreal administration, etc.)). The pharmaceutical preparation is sterile, non-pyrogenic, and nontoxic to the eye.

[0039] As used herein, the term "pharmaceutical composition" refers to a combination of one or more pharmaceutical agents and one or more inert or active carriers such that the composition is particularly suitable for in vitro, in vivo or ex vivo diagnostic or therapeutic applications. A pharmaceutical composition comprises a physical entity that is administered to a subject and can be in the form of a solid, semisolid or liquid dosage form, such as a tablet, capsule, orally disintegrating tablet, pill, powder, suppository, solution, elixir, syrup, suspension, cream, lozenge, ointment, spray, etc. A pharmaceutical composition can comprise a single pharmaceutical formulation (e.g., extended release, immediate release, delayed release, nanoparticles, etc.) or multiple formulations (e.g., immediate release and delayed release, nanoparticles and non-nanoparticles, etc.). The terms "pharmaceutical composition" and "pharmaceutical formulation" can be used interchangeably.

[0040] As used herein, the term "pharmaceutically acceptable carrier" refers to any standard pharmaceutical carrier, such as phosphate-buffered saline solution, water, emulsions (e.g., such as oil / water or water / oil emulsions), and various types of wetting agents. The composition may also contain stabilizers and preservatives. For examples of carriers, stabilizers, and adjuvants, see, e.g., Martin, Remington's Pharmaceutical Sciences, 15th Ed., Mack Publ. Co., Easton, Pa.

[1975] ; herein incorporated by reference in its entirety.

[0041] As used herein, the term "pharmaceutically acceptable salt" refers to any acid or base of a pharmaceutical agent or active metabolite or residue thereof. As known to those skilled in the art, "salts" of the compounds of the present invention may be derived from inorganic or organic acids and bases. Examples of acids include, but are not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, perchloric acid, fumaric acid, maleic acid, phosphoric acid, glycolic acid, lactic acid, salicylic acid, succinic acid, p-toluenesulfonic acid, tartaric acid, acetic acid, citric acid, methanesulfonic acid, ethanesulfonic acid, formic acid, benzoic acid, malonic acid, naphthalene-2-sulfonic acid, benzenesulfonic acid, and the like. Other acids, such as oxalic acid, although not pharmaceutically acceptable in themselves, may be used to prepare salts that serve as intermediates for obtaining the compounds of the present invention and their pharmaceutically acceptable acid addition salts.

[0042] As used herein, the term "administering" refers to the act of administering a drug, prodrug, or other agent or therapeutic treatment (e.g., a composition of the present invention) to a subject (e.g., a subject or cells, tissues, and organs in vivo, in vitro, or ex vivo). Exemplary routes of administration to humans can be via the eye (ocular), mouth (oral), skin (transdermal), nose (nasal), lung (inhalation), oral mucosa (oral), ear, rectum, by injection (e.g., intravenous, subcutaneous, intratumoral, intraperitoneal, etc.), etc.

[0043] As used herein, the term "co-administration" refers to administering at least two reagents (e.g., Compound 1 and one or more additional therapeutic agents) or therapies to a subject. In some embodiments, the co-administration of two or more reagents / therapies occurs simultaneously. In other embodiments, the co-administration of two or more reagents / therapies is sequential (e.g., a first reagent / therapy is administered before a second reagent / therapy). In some embodiments, two or more therapies are administered simultaneously, but are sequentially released (e.g., absorbed, become bioavailable, etc.). It will be appreciated by those skilled in the art that the formulation and / or route of administration of the various reagents / therapies used may vary. Those skilled in the art can easily determine a suitable co-administration dosage. In some embodiments, when co-administering a reagent / therapy, each reagent / therapy is administered with a dosage lower than that suitable for its separate administration.

[0044] Provided herein are pharmaceutical preparations of biologically active, aqueous formulations of photoreceptor-protective peptides, pharmaceutical preparations thereof, and methods and methods of preventing photoreceptor death.

[0045]

[0046] Compound 1. His-His-Ile-Tyr-Leu-Gly-Ala-Val-Asn-Tyr-Ile-Tyr-amide (SEQ ID NO: 1); Formula 1.

[0047] Some embodiments relate to the C-terminal amide peptide, Compound 1 (as above), or a pharmaceutically acceptable salt thereof. Certain embodiments relate to the polyacetate salt of Compound 1. Certain other embodiments relate to the triacetate salt of Compound 1.

[0048] These compounds can be used in pharmaceutical preparations for preventing Fas- or TRAIL-mediated apoptosis in photoreceptors of the eye. In a FasL-induced photoreceptor toxicity model, in 661W cells, IC 50 Compound 1 was 10-fold more potent than Met-12 in preventing caspase-8 activation and approximately 3-fold more potent than Met-12 at the dose that achieved maximal inhibition. In an in vivo rat model of retinal detachment, Compound 1 was at least 10-fold more potent than Met-12 in protecting photoreceptor cells from apoptosis and, unlike Met-12, could be effectively delivered in a clinically acceptable formulation.

[0049] As demonstrated in the Examples, Fas inhibition by Compound 1 resulted in significant preservation of photoreceptor cells in vivo. In 661W cells, Compound 1 treatment resulted in substantial inhibition of caspase-8 activation. Thus, it is believed that administration of Compound 1 to subjects with an ocular condition, disease, or condition or disease affecting ocular health can result in improved protection of retinal cells, including but not limited to photoreceptors, retinal pigment epithelial cells, and retinal ganglion cells, from Fas-mediated apoptosis, thereby resulting in improvement and / or treatment of the ocular condition, disease, or condition or disease affecting ocular health.

[0050] In clinical practice, patients usually have detachment that has occurred. Animal models of retinal-RPE separation show that Fas pathway activation occurs earlier and maintains high levels throughout the detachment process (Zacks et al. Arch Ophthalmol 2007; 125: 1389-1395, Zacks et al. IOVS 2004; 45 (12): 4563-4569.8.). The separation of retina and RPE is also encountered in a wide range of retinal diseases. It is expected that the clinical relevance of anti-Fas therapy in retinal cell survival is not limited to retinal detachment. For example, Fas-mediated apoptosis may play a role in photoreceptor cell death in age-related macular degeneration (AMD) (Dunaief et al. Arch Ophthalmol. 2002; 120(11):1435-1442; Zacks et al. Arch Ophthalmol 2007; Petrukhin K. New therapeutic targets in atrophicage-related macular degeneration. Expert Opin Ther Targets. 2007. 11:625–639; Miller JW. Treatment of age-related macular degeneration: beyond VEGF. Jpn JOphthalmol. 2010. 54:523–528; Rogala J, Zangerl B, Assaad N, Fletcher EL, Kalloniatis M, Nivison-Smith L. In Vivo Quantification of Retinal Changes Associated with Drusen in Age-Related Macular Degeneration. Invest Ophthalmol Vis Sci. 2015.56:1689–1700, the entire contents of which are incorporated herein by reference). Age-related macular degeneration is characterized by progressive degeneration of the RPE and causes outer retinal degeneration and reorganization similar to that occurring after retinal detachment (Jager et al. N Engl J Med. 2008; 358:2606-17, Johnson et al. Invest Ophthalmol Vis Sci. 2003; 44:4481-488, the entire contents of which are incorporated herein by reference).In the angiogenic form of AMD, there is also exudation of subretinal fluid, resulting in actual separation of the tissue from the underlying RPE (Jager et al. N Engl J Med. 2008; 358: 2606-17, the entire contents of which are incorporated herein by reference). Angiogenic AMD can lead to prolonged retinal-RPE separation and Fas pathway activation time. The utility of anti-Fas therapy is likely to be as an adjunct to protecting retinal cells (e.g., photoreceptors and retinal pigment epithelium) in treating underlying conditions (Brown et al. N Engl J Med. 2006 Oct. 5; 355 (14): 1432-44, the entire contents of which are incorporated herein by reference).

[0051] Glaucoma is a progressive degenerative eye condition characterized by the death of retinal ganglion cells (RGCs), and previously published studies have demonstrated that RGCs die by apoptosis (Ji et al. Vision Res. 2005; 45(2): 169-179). Intraocular pressure (IOP) is a major risk factor for the development of glaucoma, and efforts have been made to lower IOP using prostaglandin analogs to prevent RGC apoptosis (Doucette and Walter. Ophthalmic Genet. 2016; 12: 1-9). Fas is also involved in the death of RGCs (Gregory et al. PLoS One. 2011; 6(3): e17659), and animal models of IOP show increased expression of Fas and FasL (Ju et al. Brain Res. 2006; 1122(1): 209-221), suggesting the potential utility of Fas inhibition as a means of protecting RGC viability and alleviating the degenerative nature of glaucoma.

[0052] In some embodiments, the polypeptide can be prepared by methods known to those of ordinary skill in the art. For example, the claimed compound 1 can be synthesized using standard solid phase peptide synthesis techniques (e.g., Fmoc). Alternatively, the polypeptide can be synthesized using recombinant DNA technology (e.g., using bacterial or eukaryotic expression systems) using an overexpressed peptide and a suitable amidase to perform C-terminal amidation.

[0053] Specifically, as described in Example 1, as known to those skilled in the art, compound 1 can be obtained by: 60 HIYLGATNYIY 71 (SEQ ID NO: 2) was constructed onto an amino resin, and after deprotection and resin cleavage, its C-terminal amide H 60 HIYLGATNYIY 71-NH2, Compound 1 (SEQ ID NO: 1). Specifically, Compound 1 can conceptually be obtained from the c-Met sequence by normal amide hydrolysis between residues 59 and 60 and non-natural cleavage of the peptide chain between the peptide nitrogen and the α-carbon of residue 72 (rather than at the carbonyl carbon of residue 71). This is not a naturally occurring cleavage. Met-12 has been previously described in U.S. Patent No. 8,343,931, the entire contents of which are incorporated herein.

[0054] The use of a C-terminally amidated peptide (i.e., compound 1) is based on the belief that this particular modification can increase the pH at which the peptide is soluble in water or miscible in micelles by removing the free carboxylic acid (which is significantly deprotonated at pH greater than 3). The resulting material does not have a C-terminal anion at any physiologically relevant pH, or is a zwitterionic species under any physically relevant circumstances, and is a trivalent cationic species at pH below about 5. This change is probably most easily achieved by conversion to an amide or ester (neither of which can be deprotonated under physiological conditions). Amides are more biologically and chemically stable than esters and are less hydrophobic, so simple primary amides were chosen.

[0055] In certain embodiments, compound 1 can be produced by converting Met-12 to its C-terminal primary amide to form compound 1, although it is generally more practical to construct the peptide from the first amino acid residue that has been aminated using amino resins familiar to those skilled in the art. As described in the Examples section below, compound 1 was initially obtained and tested as the trihydrochloride salt, although the triacetate salt was later found to be more advantageous for formulation.

[0056] Compared to Met-12, the use of Compound 1 has certain advantages. Specifically, as shown in the following examples, Compound 1 can be formulated with surfactants to produce micellar solutions at pH values ​​and additive doses that are typical for ocular formulations. Secondly, based on in vitro efficacy assays, the IC 50It was determined that Compound 1 was surprisingly 10-fold more potent than Met-12, and approximately 3-fold more potent, as measured by maximum inhibitory concentration. Specifically, when Met-12 and Compound 1 were tested in vitro in the same formulation, Compound 1 had a higher dose efficacy than Met-12. This allows the same physiological effect to be achieved with a smaller amount of Compound 1 than with Met-12. Third, in in vivo testing in a rat model of retinal detachment, Compound 1 was unexpectedly at least five times more potent than Met-12 in preventing apoptosis of photoreceptor cells in the detached portion of the retina. Fourth, in some disclosed formulations of Compound 1, the efficacy achieved in the rat retinal detachment model was more than 10-fold lower than that achieved with Met-12. Finally, Compound 1 showed significantly prolonged half-lives in the vitreous humor and in the vitreous treated rabbit retina, and these half-lives could be extended to varying degrees by using different formulations, allowing the total retinal exposure to Compound 1 to be controlled by the choice of formulation.

[0057] In some embodiments, Compound 1 is effective in one or more of: preventing / inhibiting / reducing Fas-mediated photoreceptor apoptosis, preventing apoptosis of retinal pigment epithelial cells of the eye, increasing photoreceptor survival, preventing cell death associated with age-related macular degeneration (AMD), preventing cell death associated with retinal detachment, etc. In some additional embodiments, Compound 1 is effective in protecting retinal ganglion cells, which receive visual information from photoreceptors via two interneuron types: bipolar cells and retinal amacrine cells.

[0058] In some embodiments, a therapeutically effective amount of Compound 1 or a preparation thereof (i.e., a formulation or composition) is administered to a mammalian subject in need of treatment (e.g., for a specific eye condition) at a location sufficient to inhibit or attenuate apoptosis in the patient's body (e.g., within a desired tissue). Preferred subjects are humans with an eye condition, disease, or condition or disease that affects eye health.

[0059] The amount administered is sufficient to result in improved protection of retinal cells and / or retinal ganglion cells (including but not limited to photoreceptors, retinal pigment epithelium and retinal ganglion) from Fas-mediated apoptosis, or to prevent retinal cell death, thereby resulting in improvement and / or treatment of an ocular condition, disease, or condition or disease affecting ocular health.

[0060] Determination of a therapeutically effective dose is within the capabilities of those skilled in the art. In some embodiments, an effective human dose will be in the range of 5-10,000 μg / eye, 50-5,000 μg / eye, or 100-2,000 μg / eye. Repeated doses are planned to maintain effective levels (e.g., weekly, every other week, monthly, quarterly, semi-annually, etc.).

[0061] In some embodiments, the pharmaceutical preparation is a sterile, non-pyrogenic liquid and includes at least 0.1 mg / ml (e.g., >0.1, >0.2, >0.5, >0.6, >0.7, >0.8, and >0.9), at least 1 mg / ml (e.g., >1 mg / ml, >2 mg / ml, >5 mg / ml, >10 mg / ml, etc.) of a peptide / polypeptide described herein (e.g., 1 mg / ml, 2 mg / ml, 5 mg / ml, 10 mg / ml, or more of a peptide / polypeptide (e.g., Compound 1)).

[0062] In some embodiments, therapeutic dose includes at least 0.01ml (for example, 0.01ml ... 0.02ml ... 0.05ml ... 0.1ml ... 0.2ml ... 0.5ml ... 1ml ... 2ml ... 3ml ... 4ml, and volume and scope therebetween) liquid pharmaceutical preparation, the latter includes the peptide / polypeptide (for example, compound 1) of photoreceptor- or RPE-protection.In some embodiments, 10 to 500 μ l liquid volume is injected into human eyes (for example, 10 μ l, 20 μ l, 30 μ l, 40 μ l, 50 μ l, 75 μ l, 100 μ l, 200 μ l, 300 μ l, 400 μ l, 500 μ l, and volume and scope therebetween). In some embodiments, a volume of 50 to 600 μl is injected into the human eye (e.g., 50 μl, 75 μl, 100 μl, 200 μl, 300 μl, 400 μl, 500 μl, 600 μl, and volumes and ranges therebetween). In some embodiments, when injected intraoperatively, milliliter volumes can be used (e.g., up to the total volume of the vitreous cavity (e.g., about 4 ml)). In some embodiments, the compound can be incorporated into the perfusate used to maintain internal intraocular pressure during vitrectomy.

[0063] In some embodiments, a single dose is provided (e.g., to treat an acute condition (e.g., retinal detachment). In some embodiments, multiple doses are provided (e.g., daily, weekly, monthly, etc.) for the treatment of a chronic condition. The formulation may vary depending on the duration of exposure required for the condition being treated.

[0064] In some embodiments, the therapeutic dose is titrated upward from low levels to optimize safety and efficacy. In some embodiments for intravitreal injection, the dose comprises 0.01 to 5 mg of peptide (eg, 0.1 and 2.0 mg).

[0065] In some embodiments, the pharmaceutical product (i.e., formulation and / or composition) comprises one or more excipients. Excipients suitable for ophthalmic application include, but are not limited to, tonicity agents, preservatives, chelating agents, buffers, surfactants, cosolvents, and antioxidants. Suitable tonicity regulators include mannitol, sodium chloride, glycerol, sorbitol, and the like. Suitable preservatives include parabens, benzalkonium chloride, benzododecinium bromide, polyquaternium-1, and the like. Suitable chelating agents include sodium edetate, and the like. Suitable buffers include phosphates, borates, citrates, acetates, tromethamine, and the like. Suitable surfactants include ionic and nonionic surfactants, but preferably nonionic surfactants, such as polysorbates, polyethoxylated castor oil derivatives, polyethoxylated fatty acids, polyethoxylated alcohols, polyoxyethylene-polyoxypropylene block copolymers (poloxamers), and oxyethylated tert-octylphenol formaldehyde polymers (tyloxapol). Other suitable surfactants may also be included. Suitable antioxidants include sulfites, thiosulfates, ascorbates, BHA, BHT, tocopherol, and the like.

[0066] The compositions of the present invention optionally include additional active agents. These additional active agents may include anti-TNF antibodies, such as adalimumab (Ophthalmic Surg Lasers Imaging Retina 45, 332 (2014), Curr Eye Res 39, 1106 (2014)) or etanercept (PLoS One, 7, e40065) or kinase inhibitors shown to preserve retinal structure, such as ROCK inhibitor Y-27632 (Molecular Medicine Reports 12, 3655 (2015)), adenosine kinase inhibitor ABT-702 (Life Sci 93, 78 (2013)) or JNK inhibitory peptide D-JNK-1 (Diabetes 50, 77 (2001), Adv Exptl Med Biol 854, 677 (2016)) or docosahexaenoic acid (J Lipids 10, 10). Res, 54, 2236 (2013)) or the RXR pan-agonist PA024 (supra) or necrostatin or RIP kinase inhibitors such as Dabrafenib (Cell Death Dis 5, 1278 (2014)).

[0067] In some exemplary embodiments, at least one of the following may be added to the pharmaceutical preparation: an excipient such as polysorbate 20 (e.g., up to 3%), poloxamer 407 (e.g., up to 2%), tyloxapol (e.g., up to 3%), cremophor (e.g., up to 1%); and / or a cosolvent (e.g., 0.5 to 50%), such as N,N-dimethylacetamide, ethanol, PEG-400, propylene glycol, dimethyl sulfoxide (DMSO); oil or cyclodextrin.

[0068] In a further exemplary embodiment, the pharmaceutical composition may include at least one nonionic surfactant (e.g., 0.1%-20% w / w of the composition), such as polysorbate 80, polysorbate 20, poloxamer, or tyloxapol. In addition, the pharmaceutical composition may include an organic cosolvent in an amount of about 1-50%, such as propylene glycol or dimethyl sulfoxide. Alternatively, the pharmaceutical composition may include an organic cosolvent in an amount of about 1-20%, such as N,N-dimethylacetamide, ethanol, PEG-400, propylene glycol, DMSO. Alternatively, the pharmaceutical composition may include an organic cosolvent in an amount of about 1-5%, such as propylene glycol or dimethyl sulfoxide. Alternatively, the pharmaceutical composition may include an isotonic agent such as mannitol, sorbitol, glucose, or trehalose, or an inorganic salt such as sodium chloride, in an amount sufficient to bring the osmotic pressure of the composition to a range of 250-400 mOsm / L.

[0069] The pH of the composition may be in the range of 2.5-6.0. The pH may be controlled by a suitable buffer and in the range of 3.0-5.0 or 3.5-4.5.

[0070] In another exemplary embodiment, the pharmaceutical composition may include at least one nonionic surfactant (e.g., 0.5%-10% w / w of the composition), such as polysorbate 80, polysorbate 20, poloxamer, or tyloxapol. In addition, the pharmaceutical composition may include an organic cosolvent such as propylene glycol or dimethyl sulfoxide in an amount of about 1-50%. Alternatively, the pharmaceutical composition may include an organic cosolvent such as propylene glycol or dimethyl sulfoxide in an amount of about 1-20%. Alternatively, the pharmaceutical composition may include an organic cosolvent such as N,N-dimethylacetamide, ethanol, PEG-400, propylene glycol, DMSO in an amount of about 1-5%. Alternatively, the pharmaceutical composition may include an isotonic agent such as mannitol, sorbitol, glucose, or trehalose, or an inorganic salt such as sodium chloride, in an amount sufficient to bring the osmotic pressure of the composition to a range of 250-400 mOsm / L. The pH of the composition may be in the range of 2.5-6.0. The pH can be controlled by a suitable buffer and be in the range of 3.0-5.0 or 3.5-4.5.

[0071] In another exemplary embodiment, the pharmaceutical composition may include at least one nonionic surfactant (e.g., 1%-3% w / w of the composition), such as polysorbate 80, polysorbate 20, poloxamer, or tyloxapol. In addition, the pharmaceutical composition may include an organic cosolvent such as propylene glycol or dimethyl sulfoxide in an amount of about 1-50%. Alternatively, the pharmaceutical composition may include an organic cosolvent such as N,N-dimethylacetamide, ethanol, PEG-400, propylene glycol, DMSO in an amount of about 1-20%. Alternatively, the pharmaceutical composition may include an organic cosolvent such as propylene glycol or dimethyl sulfoxide in an amount of about 1-5%. Alternatively, the pharmaceutical composition may include an isotonic agent such as mannitol, sorbitol, glucose, or trehalose, or an inorganic salt such as sodium chloride, in an amount sufficient to bring the osmotic pressure of the composition to a range of 250-400 mOsm / L. The pH of the composition may be in the range of 2.5-6.0. The pH can be controlled by a suitable buffer and be in the range of 3.0-5.0 or 3.5-4.5.

[0072] In some embodiments, a pharmaceutical composition can comprise Compound 1, or a pharmaceutically acceptable salt thereof, and Poloxamer 407 (eg, 0.1-2% w / w of the composition) in an aqueous medium having a pH range of 3.0-6.0.

[0073] In some embodiments, the pharmaceutical composition may comprise Compound 1 or a pharmaceutically acceptable salt thereof and poloxamer 407 (e.g., 0.1-2% w / w of the composition) in an aqueous medium buffered with sodium propionate / propionic acid or sodium acetate / acetic acid at a pH in the range of 4.0-5.0.

[0074] In some embodiments, the pharmaceutical composition may comprise Compound 1 or a pharmaceutically acceptable salt thereof and poloxamer 407 (e.g., 0.1-2% w / w of the composition) in an aqueous medium buffered with sodium propionate / propionic acid or sodium acetate / acetic acid in the pH range of 4.0-5.0, made isotonic with 3-5% mannitol.

[0075] In some further embodiments, the pharmaceutical composition may comprise Compound 1 or a pharmaceutically acceptable salt thereof and polysorbate 20 (e.g., 0.1-3% w / w of the composition), propylene glycol (e.g., 3% w / w of the composition) in an aqueous medium having a pH range of 3.0-6.0.

[0076] In certain further embodiments, the pharmaceutical composition may comprise Compound 1, or a pharmaceutically acceptable salt thereof, and polysorbate 20 (e.g., 0.1-3% w / w of the composition), propylene glycol (e.g., 3% w / w of the composition), in a sodium acetate / acetic acid buffered aqueous medium having a pH in the range of 4.0-5.0.

[0077] In some further embodiments, the pharmaceutical composition may comprise Compound 1, or a pharmaceutically acceptable salt thereof, and polysorbate 20 (e.g., 0.1-3% w / w of the composition) and mannitol (e.g., 3-5% w / w of the composition) in an aqueous medium having a pH range of 3.0-6.0.

[0078] In certain further embodiments, the pharmaceutical composition may comprise Compound 1, or a pharmaceutically acceptable salt thereof, and polysorbate 20 (e.g., 0.1-3% w / w of the composition) and mannitol (e.g., 3-5% w / w of the composition) in a sodium acetate / acetic acid buffered aqueous medium having a pH in the range of 4.0-5.0.

[0079] In some embodiments, the pharmaceutical composition may comprise Compound 1, or a pharmaceutically acceptable salt thereof, and poloxamer 407 (e.g., 0.1-2% w / w of the composition) and polysorbate 20 (e.g., 0.1-2% w / w of the composition) in an aqueous medium having a pH range of 3.0-6.0.

[0080] In some embodiments, the pharmaceutical composition may comprise Compound 1, or a pharmaceutically acceptable salt thereof, and poloxamer 407 (e.g., 0.1-2% w / w of the composition) and polysorbate 20 (e.g., 0.1-2% w / w of the composition) in an aqueous medium buffered with sodium propionate / propionic acid or sodium acetate / acetic acid at a pH in the range of 4.0-5.0.

[0081] In some embodiments, the pharmaceutical composition may comprise Compound 1, or a pharmaceutically acceptable salt thereof, and poloxamer 407 (e.g., 0.1-2% w / w of the composition) and polysorbate 20 (e.g., 0.1-2% w / w of the composition) in an aqueous medium buffered with sodium propionate / propionic acid or sodium acetate / acetic acid at a pH in the range of 4.0-5.0 and made isotonic with 3-5% mannitol.

[0082] In some embodiments, the pharmaceutical compositions described above may comprise Compound 1, but without chloride as a counterion, with acetate being a preferred alternative. Such compositions may exhibit properties superior to those containing chloride ions.

[0083] In some embodiments, the weight ratio of the peptide / polypeptide (e.g., Compound 1) relative to the weight of the non-aqueous excipient in the pharmaceutical formulation is 1%-25%, which in turn is 0.1-20% of the excipient, such as poloxamer, polysorbate 20, propylene glycol, and mannitol.

[0084] The weight ratio of the peptide / polypeptide (eg, Compound 1) relative to the weight of the pharmaceutical formulation can be at least about 0.1%, at least 0.5%, at least 1%, at least about 2%, at least about 3%.

[0085] The following two exemplary compositions, with the amounts of each ingredient within the ranges shown, provide two of several compositions useful for treating or preventing various ocular diseases or conditions (e.g., retinal) or preventing retinal cell death in a subject resulting from an ocular disease or condition, or the like:

[0086] Exemplary Formulation 1:

[0087]

[0088]

[0089] Exemplary Formulation II:

[0090] Compound 1 triacetate 0.1-2 mg / mL Polysorbate 20 0.1-1.0％ Additives (eg, mannitol) 2.5-5％ Acetic acid 10mM NaOH Until pH>3 Water(WFI) Until 100%

[0091] In some embodiments, the compositions of the invention are administered ophthalmically, for example, using the techniques described herein and / or other techniques known to those of skill in the art (e.g., injection, topical administration, etc.) (see, e.g., Janoria et al., Expert Opin Drug Deliv., 4(4):371-388 (July 2007); Ghate & Edelhauser, Expert Opin Drug Deliv., 3(2):275-87 (2006); Bourges et al., Adv Drug Deliv Rev., 58(11):1182-202 (2006), Epub 2006 Sep. 22; Gomes Dos Santos et al., Curr Pharm Biotechnol., 6(1):7-15 (2005); herein incorporated by reference in their entireties). The compositions can be administered using any method known to those of ordinary skill in the art. Non-limiting examples include topical, subconjunctival, subtenon, intravitreal, subretinal, or injected into the anterior chamber of the subject's eye. Other modes of administration include systemic administration, including intravenous administration and oral administration.In certain embodiments, the composition is administered intravitreally.

[0092] Certain embodiments relate to pharmaceutical compositions comprising a Compound 1 polypeptide and a pharmaceutically acceptable carrier. Any carrier that can deliver the polypeptide without destroying the vector within the carrier is a suitable carrier, and such carriers are well known in the art.

[0093] Composition can be formulated and packaged to be suitable for parenteral, oral or body surface administration. For example, parenteral preparations can be sterile, non-pyrogenic products, and can be composed of quick-release or delayed-release liquid products, dry powders, emulsions, suspensions or any other standard formulations. The oral formulations of the pharmaceutical composition can be, for example, liquid solutions, such as compositions of an effective amount dissolved in diluents (e.g., water, saline, fruit juice, etc.), suspensions in suitable liquids or suitable emulsions. Oral formulations can also be delivered in tablet form, and can include excipients, colorants, diluents, buffers, wetting agents, preservatives, flavorings and pharmacologically compatible excipients. Body surface preparations can include compounds that enhance the absorption or penetration of active ingredients by the skin or other affected areas, such as dimethyl sulfoxide and related analogs. Pharmaceutical compositions can also be delivered using transdermal devices, such as patches, which can include compositions in suitable solvent systems with adhesive systems, such as acrylic emulsions and polyester patches. Sterile compositions can be delivered via eye drops or other body surface eye delivery methods. Sterile, non-pyrogenic compositions can be delivered intraocularly anywhere in the eye, including, for example, the vitreous cavity, the anterior chamber, and the like. Sterile, non-pyrogenic compositions can be delivered intravitreally, as is commonly done, such as intravitreal injections of Lucentis (ranabizumab), Avastin (bevazizumab), triamcinolone acetonide, antibiotics, and the like. The composition can be delivered periocularly (e.g., to tissue surrounding the eyeball (sphere), but within the bony orbit). The composition can be delivered via an intraocular implant (e.g., ganciclovir implant, fluocinolone acetonide implant, etc.). In intraocular implant delivery, a device containing the composition of the present invention is surgically implanted (e.g., in the vitreous cavity) and the drug is released into the eye (e.g., at a predetermined rate). The composition can be administered using encapsulated cell technology (e.g., from Neurotech), in which genetically modified cells are engineered to produce and secrete a composition comprising a compound 1 polypeptide. The compositions can be delivered via transscleral drug delivery using sutures or a device placed alongside the globe, which will slowly elute the drug and then diffuse it into the eye.

[0094] Some embodiments relate to compositions, test kits, systems and / or methods for preventing, suppressing, blocking and / or reducing photoreceptor, RPE cell or retinal ganglion cell death.Some embodiments relate to suppressing the apoptosis of photoreceptor.Some embodiments relate to suppressing the apoptosis in the cell of the retinal pigment epithelium of eyes.Some embodiments relate to suppressing the apoptosis in the retinal ganglion cells of eyes.In some embodiments, photoreceptor death and / or apoptosis and / or retinal pigment epithelium apoptosis and / or apoptosis and / or retinal ganglion cell apoptosis and / or death are caused by retinal detachment, age-related macular degeneration, glaucoma, wound, cancer, tumor, inflammation, uveitis, diabetes, hereditary retinal degeneration and / or the disease that affects photoreceptor cell, abnormal retinal pigment epithelium or retinal ganglion.

[0095] In some embodiments, the present invention enhances photoreceptor, RPE, or retinal ganglion cell viability and / or inhibits photoreceptor death (e.g., during retinal detachment and / or in ocular conditions not involving retinal detachment.

[0096] In some embodiments, the present invention can be used to enhance photoreceptor, RPE or retinal ganglion cell viability and / or inhibit photoreceptor, RPE or retinal ganglion cell death in a variety of conditions and / or diseases, including, but not limited to, macular degeneration (e.g., dry, wet, non-exudative or exudative / neoangiogenic), ocular tumors, glaucoma, inherited retinal degenerations (e.g., retinitis pigmentosa, Stargardt disease, Usher syndrome, etc.), ocular inflammatory diseases (e.g., uveitis), ocular infections (e.g., bacterial, fungal, viral), autoimmune retinitis (e.g., triggered by infection), trauma, diabetic retinopathy, choroidal neovascularization, retinal ischemia, retinal vascular occlusive diseases (e.g., branch retinal vein occlusion, central retinal vein occlusion, branch retinal artery occlusion, central retinal artery occlusion, etc.), pathological myopia, angioid streaks, macular edema (e.g., of any etiology), central serous chorioretinopathy.

[0097] Some embodiments relate to administering compositions to suppress photoreceptor, RPE or retinal ganglion cell death (for example, apoptosis).In some embodiments, compositions comprises medicine, small molecule, peptide, nucleic acid, molecular complex etc.In some embodiments, the invention provides the administration of photoreceptor, RPE or retinal ganglion cell protective polypeptide to suppress photoreceptor or RPE or retinal ganglion cell apoptosis.

[0098] Some embodiments relate to methods of using polypeptides to reduce activation of one or more members of the TNFR superfamily (Fas or TRAIL, which is desired in photoreceptors and / or the retina). In some embodiments, this method is used, for example, to inhibit cell death (e.g., apoptosis) in cells and tissues and can be used in vivo, ex vivo, or in vitro. Thus, according to such methods, Compound 1 can be used to reduce cell death (e.g., retinal cell death). For in vitro applications, Compound 1 can be provided to cells, typically cell populations (e.g., in a suitable preparation such as a buffer solution), in an amount and time course sufficient to inhibit intracellular apoptosis and inhibit inflammation. If desired, a control population not treated with the polypeptide of the invention can be observed to confirm the inhibitory effect of the polypeptide of the invention on reducing cell death or inflammation in similar cell populations.

[0099] In some embodiments, provided herein are methods of treating or preventing retinal cell death resulting from a variety of ocular diseases or conditions (e.g., retinal), including: glaucoma, maculopathy / retinal degeneration, such as macular degeneration, including age-related macular degeneration (AMD), such as non-exudative age-related macular degeneration and exudative age-related macular degeneration; choroidal neovascularization; retinopathy, including diabetic retinopathy, acute and chronic macular neuroretinopathy, central serous chorioretinopathy; and macular edema, including cystoid macular edema, diabetic macular edema; uveitis / retinitis / choroiditis, such as acute multifocal squamous pigment epitheliopathy, Behcet's disease, and retinal edema. disease), retinochoroidal disease, infectious (syphilis, Lyme disease, tuberculosis, toxoplasmosis), uveitis, including intermediate uveitis (pars planitis) and anterior uveitis, multifocal choroiditis, multiple transient white dot syndrome (MEWDS), ocular sarcoidosis, posterior scleritis, creeping choroiditis, subretinal fibrosis, uveitis syndrome, and Vogt-Koyanagi-Harada syndrome; vascular disease / exudative disease, such as retinal artery occlusive disease, central retinal vein occlusion, disseminated intravascular coagulopathy, branch retinal vein occlusion, hypertensive fundus changes, ocular ischemic syndrome, retinal artery microaneurysms, outer exudative retinopathy retinal vascular occlusion, parafoveal telangiectasia, hemiretinal vein occlusion, optic disc phlebitis, central retinal artery occlusion, branch retinal artery occlusion, carotid artery disease (CAD), frosty dendritic vasculitis, sickle cell retinopathy and other hemoglobinopathies, angioid streaks, familial exudative vitreoretinopathy, Eales' disease, traumatic / surgical conditions (sympathetic ophthalmia, uveitic retinopathy, retinal detachment, trauma, laser, PDT, photocoagulation, hypoperfusion during surgery), radiation retinopathy, bone marrow transplant retinopathy; proliferative diseases such as proliferative vitreoretinopathy and epiretinal membranes, proliferative diabetic retinopathy.Infectious diseases: ocular histoplasmosis, ocular toxocariasis, ocular histoplasmosis syndrome (OHS), endophthalmitis, toxoplasmosis, retinopathy associated with HIV infection, choroidopathy associated with HIV infection, uveopathy associated with HIV infection, viral retinitis, acute retinal necrosis, progressive outer retinal necrosis, fungal retinal disease, ocular syphilis, ocular tuberculosis, diffuse unilateral subacute neuroretinitis, and myiasis; genetic diseases such as retinitis pigmentosa, systemic disorders associated with retinal dystrophy, congenital stationary night blindness, cone dystrophy, Stargardt disease, fundus macularis, congenital macular degeneration, pattern dystrophy of the retinal pigment epithelium, X-linked retinoschisis, Sorsby's fundus dystrophy, benign concentric maculopathy, Bietti lens dystrophy, pseudoxanthoma elasticum. Retinal tears / holes: retinal detachment, macular hole, giant retinal tear; tumors, such as tumor-related retinopathy, congenital hypertrophy of the RPE, posterior uveal melanoma, choroidal hemangioma, choroidal osteoma, choroidal metastatic carcinoma, combined hamartoma of the retina and retinal pigment epithelium, retinoblastoma, fundus vascular proliferative tumors, retinal astrocytoma, intraocular lymphoid tumors; and other diseases and conditions, such as punctate inner choroidopathy, acute posterior multifocal squamous pigment epitheliopathy, myopic retinal degeneration, acute retinal pigment epitheliitis, corneal dystrophy or developmental abnormalities, etc.

[0100] Some embodiments provide a method for increasing photoreceptor, RPE or retinal ganglion survival, which includes administering a pharmaceutical composition comprising compound 1 or its pharmaceutically acceptable salt. According to good pharmaceutical practice, the pharmaceutical compound can be administered in the form of a composition formulated with a pharmaceutically acceptable carrier and optional excipients, adjuvants, etc. The composition can be in the form of a solid, semisolid or liquid dosage form: such as powders, solutions, elixirs, syrups, suspensions, creams, drops, ointments and sprays. As will be appreciated by those skilled in the art, depending on the selected route of administration (such as eye drops, injections, etc.), the composition form is determined. In general, it is preferred to use the sterile unit dosage form of the inhibitor of the present invention, to achieve simple and accurate administration of the active pharmaceutical compound. Typically, the therapeutically effective pharmaceutical compound exists in such dosage form, and its concentration range is about 0.01% to about 1.0% of the total composition weight, i.e., exists in an amount sufficient to provide the required unit dose.

[0101] In some embodiments, the pharmaceutical composition can be administered in a single dose or multiple doses. The specific route of administration, product requirements, and dosage regimen will be determined by a skilled person based on the condition of the individual to be treated and the individual's response to treatment. In some embodiments, a composition in unit dose form for administration to a subject comprises a pharmaceutical compound and one or more non-toxic pharmaceutically acceptable carriers, adjuvants, or vehicles. As described above, the amount of active ingredient that can be combined with these materials to produce a single dose form will vary depending on various factors. As available in the pharmaceutical field, various materials can be used as carriers, adjuvants, and vehicles in the compositions of the present invention. Injectable products such as oily solutions, suspensions, or emulsions can be formulated as needed using suitable dispersants or wetting agents and suspending agents, as known in the art. Sterile injectable products can use non-toxic parenteral-acceptable diluents or solvents, such as sterile non-pyrogenic water or 1,3-butanediol. Other acceptable vehicles and solvents that can be used can be 5% dextrose injection, Ringer's injection, and isotonic sodium chloride injection (as described in USP / NF). In addition, sterile fixed oils can be conventionally used as solvents or suspending media. For this purpose, any bland fixed oil may be employed including synthetic mono-, di-, or triglycerides. Fatty acids such as oleic acid may also be used in the preparation of injectable compositions.

[0102] There are several possible routes for drug delivery to ocular tissue. The route of administration depends on the target tissue. In certain embodiments, the route of administration can be a conventional route of administration, such as topical or systemic administration. Topical administration, primarily in the form of eye drops, can be used to treat conditions affecting the anterior segment of the eye. It can also be administered via direct injection, such as intravitreal injection, which involves injecting a drug solution directly into the vitreous humor (VH) using, for example, a 30G needle. Other routes of administration, such as using a drug carrier, may also be suitable.

[0103] In some embodiments, the pharmaceutical composition can be administered ophthalmically (i.e., to the eye), for example, using the techniques described herein, and / or other techniques known in the art (e.g., injection, topical administration, etc.) (see, Janoria et al. Expert Opinion on Drug Delivery. July 2007, Vol. 4, No. 4, Pages 371-388; Ghate & Edelhauser. Expert Opin Drug Deliv. 2006 March; 3(2): 275-87; Bourges et al. Adv Drug Deliv Rev. 2006 Nov. 15; 58(11): 1182-202. Epub 2006 Sep. 22; Gomes Dos Santos et al. Curr Pharm Biotechnol. 2005 February; 6(1): 7-15; the entire contents of which are incorporated herein by reference).

[0104] In some embodiments, the compositions can be co-administered with one or more other agents effective to protect photoreceptors and / or inhibit apoptosis.

[0105] In some embodiments, a kit comprising Compound 1 or its pharmaceutical product is provided. In some embodiments, the kit also provides devices, materials, buffers, controls, instructions, containers (e.g., vials, syringes), etc. (e.g., for administration). For example, any of the above-mentioned compositions and / or preparations can be packaged. Any of the above-mentioned compositions and preparations can be distributed in prefilled syringes. The composition and processing produce a sterile, non-pyrogenic product. The function of the packaging is to maintain the sterility of the product.

[0106] Example

[0107] During the development of the described embodiments, experiments were conducted to develop a bioactive pharmaceutical formulation of Compound 1 (e.g., for intravitreal administration). The photoreceptor protective properties of Compound 1 were tested in vitro and in vivo based on the dose of the peptide solution in DMSO. Compound 1 has poor water solubility above pH about 3 and is very prone to forming gels or precipitates in aqueous environments. Based on the intra-species vitreous volume, the effective dose was adjusted from rats to humans, and a target concentration of 10-20 mg / mL was determined as an initial target. Given the surprisingly excellent efficacy and exposure of the described embodiments as demonstrated by testing, lower concentrations (0.5-2.0 mg / mL) are more desirable (Examples 1-6).

[0108] Example 1: Compound 1 Preparation and Testing

[0109] Compound 1 peptide (peptide His-His-Ile-Tyr-Leu-Gly-Ala-Val-Asn-Tyr-Ile-Tyr-NH2; SEQ ID NO: 1) was synthesized on Fmoc-amide-AMS resin (from multiple suppliers) via Fmoc chemistry. Fmoc-protected amino acids were purchased from GL Biochem. Reagents used for coupling and cleavage were purchased from Aldrich. Solvents were purchased from Fisher Scientific.

[0110] Peptide chains were assembled on resin by iteratively removing the Fmoc protecting group and coupling the protected amino acid. DIC and HOBt were used as coupling agents, with NMM as the substrate; 20% piperidine in DMF was used as the Fmoc removal reagent. A ninhydrin test was performed after each coupling to check coupling efficiency.

[0111] After the last coupling, the resin was washed and dried, and the peptide was cleaved from the resin by treatment with a cleavage mixture (TFA / Tis / H2O / DOTA: 95 / 3 / 2 / 2). The peptide was precipitated from cold ether and collected by filtration to give 13 g of a crude product with a purity of 46% (yield: 127%).

[0112] For each of the two preparative purification runs, approximately 4.4 g of crude peptide was purified by passing through a 2-inch polymer column containing TFA buffer (buffer A, 0.1% TFA in water; buffer B, 100% acetonitrile), and the fractions with a purity of >85% were further purified using a 2-inch C18 column containing TFA buffer. The collected fractions with a purity of >95% were lyophilized to dryness, and 3.68 g of TFA salt material with a purity of >95% was obtained from 8.8 g of crude product. Sufficient aqueous HCl was added to 1.5 g of peptide (TFA as a counterion) to dissolve the peptide. The peptide in aqueous HCl was lyophilized to dryness. 1.4 g of the final peptide was obtained as an HCl salt with a purity of 97.0%. HPLC, 15% CAN in water, 0.1% TFA, Venusil XBP-C18 4.6×250 mm 1.0 mL / min; RT 17.79 min. Mass spectrometry APCI MH + 1461.5.

[0113] Microanalysis. Found: C, 52.21; H, 6.49; N, 15.42; Cl, ​​6.73. KF, 3.75%. 71 H 100 N 18 O 16 Calculated for 3HCl·3.4H2O: C, 52.24; H, 6.59; N, 15.45; Cl, ​​6.52. KF, 3.75%. % activity = 89.55%.

[0114] Later samples of the peptide were still synthesized as the trifluoroacetate salt, but anion exchange was performed with acetate to give compound 1 as the triacetate salt.

[0115] Example 2: pH-solubility curve of compound 1

[0116] As described in Example 1, Compound 1 was obtained as a trihydrochloride salt and screened for aqueous solubility at various pH values ​​by performing pH titration according to the following protocol. In some cases, Met-12 was run through the same experimental procedure to determine its pH solubility profile under the same conditions. Previous experiments failed to identify any conditions under which Met-12 could be satisfactorily formulated in a basic aqueous medium at any pH above 2.7.

[0117] Compound 1 (10 mg) was dissolved in water (270-900 μL) in a 2 mL clear plastic centrifuge tube with a vortex to produce a solution with a pH of approximately 2.4. In all cases, the peptide formed a clear solution, indicating a solubility of at least 40 mg / mL at low pH. The solution was then diluted with an appropriate amount of cosolvent or other excipient (sugar, surfactant, etc.) to produce a clear acidic solution of 10 mg of compound 1 in 900 μL of test solution at room temperature (22-23°C). A small amount of alkaline solution (usually 1.0 M or 0.1 M sodium hydroxide, but other bases are sometimes used when studying buffers) was added using a microsyringe. Between additions, the solution was mixed by vortexing and gel formation was detected by visual inspection of the solution for various types of precipitates, turbidity (as a possible sign of microprecipitates), and viscosity. pH measurements were performed at all these observation points. Some experiments titrated from the endogenous low pH value to pH 10, but subsequent titrations were not performed much above pH 7, or sometimes even lower.

[0118] Titration of aqueous Compound 1 solutions with sodium hydroxide showed slightly better pH-limited solubility than Met-12, with the pH of the clear mobile solution being approximately 3.3, compared to 3.0 for Met-12. However, when titration was performed using five buffer bases, Tris, histidine, sodium citrate, sodium borate, and sodium phosphate, instead of sodium hydroxide, viscosity and signs of aggregation were generally observed in the pH range of 2.6-2.9. In one or two cases, fibril formation was also observed below pH 3. From these experiments, it appears that Compound 1 does not have a better water solubility-pH profile than Met-12.

[0119] Example 3: pH-dependent solubility of Compound 1 in co-solvent mixtures

[0120] The pH-dependent solubility of compound 1 was examined using cosolvents and additives and compared with the solubility of Met-12 under the same conditions.

[0121] The 70% DMSO experiment was similar to the Met-12 titration in that a gel formed around pH 5.5, but in this case, likely because the C-terminus could not be ionized, the gel no longer dissolved at higher pH values.

[0122] Propylene glycol (PG) at 70% improved the solubility of Compound 1 compared to Met-12, with gelation not occurring until around pH 4.7 compared to pH 3.2 for Met-12, and then remaining as a gel to pH 10. The titration was repeated with lower amounts of PG (35%, 10%), but no improved solubility over that in pure water appeared to be observed.

[0123] A 70% PEG400 and 70% glycerol solution did not seem to work, nor did two sugar additives, 10% mannitol or 10% trehalose.

[0124] From these experiments it can be concluded that propylene glycol may be a useful cosolvent for compound 1 under some restrictive conditions, but not for Met-12.

[0125] Example 4: pH-dependent solubility of Compound 1 in a mixture of nonionic surfactants

[0126] Surprisingly, some of the surfactants examined provided significantly improved pH-solubility profiles for Compound 1, whereas none of the surfactants tested improved the pH-solubility profile for Met-12. Compound 1 in the presence of 10% tyloxapol remained clear and had acceptable viscosity until pH above 5.87. At 10% polysorbate 80, the clear solution did not become noticeably viscous until pH values ​​above 6.36. At 10% polysorbate 20, fibrils were observed at pH 3.2, but there was no sign of turbidity or gelation until pH 7.14. 10% poloxamer 407 left some ambiguity as to where insolubility might have begun, as a second phase clearly existed in the pH 5-9 range, although the solution appeared to be mobile. This appeared to consist of very large, transparent spheres that formed in solution. This is believed to be an artifact of the high poloxamer concentration, as a 15% poloxamer solution completely gelled at 27°C, while a 10% poloxamer solution showed no noticeable gelation at 25°C. However, various additives can raise or lower the sol-gel critical temperature, and conventional gel viscosity measurements do not effectively capture the initial appearance of a separate gel phase. Therefore, it is believed that there is no loss of peptide solubility in the mixture, but rather that the large amount of poloxamer forms two poloxamer phases, a sol phase and a gel phase. Povidone K30 produces a viscous solution at pH 3.60, which gels above pH 4.0.

[0127] The surfactant was then titrated relative to the amount of surfactant added. When polysorbate 20 was titrated to 3% and 1% concentrations, fibril formation was observed below pH 2.5, but in both cases no further signs of precipitation were seen until the pH reached 4.14 and 3.76, respectively. 4% poloxamer 407 produced a small and apparently non-increasing number of fibrils at the start of the titration, but no further signs of precipitation were seen until the pH reached above 6.2, and 2% poloxamer 407 produced a clear solution until the pH reached above 5.6. Once the titration began, 0.5% fibrils were seen in the solution, but no further signs of precipitation were seen until the pH reached above 4.5.

[0128] Compound 1 clearly outperformed Met-12 among most of the surfactants examined, particularly polysorbate 80, poloxamer 407, and tyloxapol, with data for polysorbate 20 somewhat ambiguous due to initial fibril formation observed, although it was clearer than most of the compounds and was a solution at pH 3-6, in contrast to Met-12 in the same vehicle. Therefore, the solubility of Compound 1 was observed in cosolvent-surfactant mixtures, starting at high additive concentrations and then at lower concentrations of one or both additives in a sparse population matrix experimental design.

[0129] Example 5: Mixed Nonionic Surfactant / Cosolvent Solubility Study of Compound 1

[0130] The combination of 70% PG and 10% polysorbate 80 produced a clear solution that became viscous at pH 3.4 and gelled at pH 5.25, making it no better than 70% PG and worse than 10% PS-80.

[0131] For 70% PG, 3% PS, viscosity began to set in at pH 4.6, but the material was still a gel at pH 5.25.

[0132] For 35% PG and 3% PS-80, fibrils appeared in solution at pH as low as 2.66, and aggregates were seen in solution at pH 3.48.

[0133] 35% PG, 10% PS-80 showed no signs of fibrils or aggregates, and significant viscosity was observed at pH 4.05, with gelation at pH 5.71 (less than 10% PS-80 itself).

[0134] 10% PG and 10% PS-80 produced clear solutions with low viscosity up to pH 4.94, and above pH 5.13 material began to precipitate.

[0135] 10% PG and 3% PS-80 produced clear solutions up to pH 3.16, but some precipitation occurred at pH 3.4.

[0136] As before, solutions containing 10% polysorbate 20 appeared to give clear mobile solutions up to pH 7, but some fibrils were visible even at low pH and tended to increase with increasing pH.

[0137] Surprisingly, the combination of 10% PG and 10% polysorbate 20 resulted in good solubility, with the onset of noticeable viscosity occurring only reproducibly above pH 7, without any visible signs of precipitation. However, after standing overnight, the solution gelled, with the pH dropping by approximately 0.2 units. Gentle stirring converted the gel into an injectable liquid.

[0138] 3% polysorbate 20 and 10% PG produced clear mobile solutions up to pH 5.3, but reducing PS-20 to 1% produced fibrils below pH 3.

[0139] 10% PG was added to the 4%, 2%, and 0.5% poloxamer formulations. The 4% poloxamer formulation appeared slightly improved, with no visible fibrils at low pH and a clear solution until at least pH 5.36. The 2% poloxamer and 10% PG formulations performed just as well as clear, mobile solutions until pH 5.74. The 0.5% poloxamer formulation showed fibrils at the start of the titration and some turbidity at pH 3.45, but maintained a low viscosity until pH 6.

[0140] PG was added to 2%, 1%, and 0.5% poloxamer formulations at 3%, 1%, or no PG, and after standing at room temperature for 3 days, stability was measured at pH 4.5, pH 5.5, and pH 7.0 using both visual and filtration analysis (see Examples below). As can be seen, fibrils were observed less frequently in fresh samples, being more likely to be observed in the presence of less excipient and at higher pH, while all pH 7.0 samples were initially turbid, and some had a noticeable precipitate. None of the pH 4.0 samples were initially turbid, with only the 0.5% PX, 0% PG sample showing slight turbidity after standing. For the pH 5.5 samples, initial slight turbidity was observed in all 0% PG samples and the 1% PG, 0.5% PX sample, but only the lowest excipient sample (0.5% PX, 0% PG) showed slight turbidity after standing. Some of these samples became slightly turbid upon stirring. Filtration analysis paints a very different picture. Using the 2% poloxamer formulation, all three pH 4 formulations had >98% recovery after filtration, and >96% recovery at pH 5.5, and still 86-93% recovery at pH 7.0. In the 1% PX formulations at pH 4, post-filtration recoveries were 97-98%, 87-93% at pH 5.5, but only 1-13% at pH 7. In the 0.5% PX formulations, post-filtration recoveries were 73-88% at pH 4, 12-43% at pH 5.5, and no recoveries were found in any of the three samples at pH 7.

[0141] From these experiments, it can be concluded that relatively low amounts of PG as a cosolvent can be modestly helpful for some surfactants, but high concentrations are detrimental, and the predictability of these formulations is poor. In PX formulations, both the amount of PX present and the formulation pH are more important than the PG level. Furthermore, visual readings do not necessarily correlate with more reliable filtration assays, and the observation of fibrils, in particular, does not appear to correlate with the amount of filterable drug present.

[0142] Example 6: Solubility study of compound 1 at lower concentrations of nonionic surfactants

[0143] In vitro and subsequent in vivo efficacy experiments demonstrated that compound 1 was 3- to 10-fold more potent in blocking FasL-induced (or retinal detachment-induced) apoptosis in photoreceptor cells. These surprising results allowed the planned human dose to be lowered to a range of 25-200 μg / eye, which reduced the maximum required concentration of the formulated drug to 2.0 mg / mL. Further experiments were conducted to identify optimal formulation conditions at this concentration, with some experiments considering even lower drug concentrations. This work considered poloxamer 407 or polysorbate 20 as surfactants. Visible turbidity and visual estimation of viscosity are both useful screening observations, but as mentioned above, they have been found not always to indicate the presence of aggregated peptides, and most subsequent work has assessed solubility by measuring the amount of drug present in samples before and after passage through 0.2-μm PVDF membranes or PALL 25mm 0.2-μm Ultipor nylon 6,6 filters. Turbid or highly viscous solutions are often difficult or impossible to filter, and when they can be filtered, they often give very low drug recoveries. Surprisingly, some mobile, clear solutions also exhibit substantial losses upon filtration, so satisfactory formulations are defined as those that give >90% drug recovery after filtration. It should be noted that solubility measurements for all fibril-forming compounds such as Compound 1 measure only kinetic solubility. Fibril formation can be very slow under many conditions, and solubility can be measured for metastable solutions, where true thermodynamic solubility relative to the most stable possible fibril form may take days to years to fully achieve. However, formulations are typically allowed to stand for 24–72 hours prior to filtration to avoid rapid precipitation, at least after formulation.

[0144] Initial experiments examined 1 mg / mL solutions of compound 1 in 3% PS-20 / 3% PG and 2% PX / 2% PG at pH 4. All yielded clear solutions, with no loss of API upon filtration. Subsequent experiments examined 2 mg / mL solutions in 2% PG with 0.1%, 0.25%, and 0.5% PX, as well as 0.5% PG with PX, at pH 3, pH 4, pH 5.5, and pH 7. With the exception of the pH 7 sample with 0.1% PX, which was slightly turbid and mobile in appearance, all other samples were clear and mobile, but no recovery occurred upon filtration. Recovery was only 78% at pH 5.5 and 92% at pH 4.0. Higher PX amounts resulted in complete recovery at pH 3 and 4.0, 93-97% at pH 5.3, and 88-92% at pH 7.0. High and low PG, 0.5% PX, were essentially identical, suggesting that PG is not very important in this area of ​​formulation manifold.

[0145] Because significant pH shifts were occasionally observed during extended storage in these experiments, similar experiments were performed using 2.0 mg / mL Compound 1 and 0.25% PX. 2% PX was run at pH 3, 4, 5.5, and 7, and analyzed by filtration assay, comparing the self-buffered material (hydrochloride titrated with NaOH) to 10 mM histidine, acetate, and citrate buffers. Acetate buffer performed at least as well as the self-buffered material at all pHs, histidine buffer performed slightly worse, and citrate buffer performed significantly worse at all three higher pHs, recovering only 75% at pH 4, compared to 91% for histidine buffer, 92% for no buffer, and 97% for acetate buffer. Acetate buffer was then standardized.

[0146] To examine the effect of isotonicity, 10 mM acetate buffer solutions at pH 4 and pH 5.5 containing 2 mg / mL Compound 1 and 0.25% PX were tested using 0.5% and 2% PG, 4.5% mannitol, 2% glycerol, and 0.8% aqueous sodium chloride. With the exception of the sodium chloride sample, all samples had recoveries of 98-99% at pH 4.0 and 90-94% at pH 5.5, while recoveries for the sodium chloride sample were 89% and 79% at the two pH values, respectively. With the exception of 0.5% PG, which was quite hypotonic, all other samples were within the 230-310 mOsm / L range. Using 10 mM acetate buffer and 4.5% mannitol as the isotonicity, five surfactant conditions were observed for 2 mg / mL Compound 1 at pH 4.0 and 5.5. These were no surfactant, 0.1% PS-20, 0.1% PS-20 and 0.25% PX, 0.25% PX, and 0.4% PX. No surfactant filtered 0% at high pH and 23% at pH 4.0, while 0.4% PX and the 0.25% PX / 0.1% PS-20 mixture recovered completely at pH 4.5 and 95% and 91%, respectively, at pH 5.5. At pH 4 and 5.5, 0.25% PX performed poorly at 96% and 91%, respectively, while 0.1% PS performed quite poorly at 90% and 65%, respectively.

[0147] Since isotonicity experiments had indicated that the solubility of the hydrochloride salt might be poor, experiments were performed using the triacetate salt of compound 1 at 2 mg / mL. Although the samples were dissolved in 10 mM acetic acid, 4.5% mannitol, and 0.4%, 0.5%, 0.6%, 0.8%, and 1.0% PX or 0.4%, 0.5%, 0.75%, 1.0%, and 1.5% PS-20, and the pH was adjusted to 4.0 or 5.5, the inherent pH of the salt at 2 mg / mL in water is 3.4-3.6 due to the weak acidity of acetic acid. All samples without pH adjustment (3.4-3.6) showed >97% recovery after filtration, and >98% at pH 4. At pH 5.5, 0.4% and 0.5% PX had 96% recovery, and higher PX concentrations produced 98-99%, while PS-20 formulations all recovered 92-94% at this pH. From these experiments, optimized formulations may contain less PX than similar PS-20-based formulations, but PS-20 will still be acceptable and may avoid some potential problems with PX, even when administered at higher concentrations.

[0148] Example 7: In vitro efficacy of compound 1

[0149] Cell culture. The 661W photoreceptor cell line was generously provided by Muayyad Al-Ubaidi (Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK). The 661W cell line is a photoreceptor line that has been immortalized by expression of the SV40-T antigen under the control of the human interphotoreceptor retinoid binding protein (IRBP) promoter (AI-Ubaidi et al., J Cell Biol., 119(6):1681-1687 (1992), incorporated herein by reference in its entirety). 661W cells express cone photoreceptor markers, including blue and green cone pigments, transducin, and cone arrestin (Tan et al., Invest Ophthalmol Vis Sci., (3): 764-768 (2004), incorporated herein by reference in its entirety), and can undergo caspase-mediated cell death (Kanan et al., Invest Ophthalmol Vis Sci., 48(1): 40-51 (2007), incorporated herein by reference in its entirety).

[0150] The 661W cell line was maintained in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, 300 mg / L glutamine, 32 mg / L putrescine, 40 μL / L β-mercaptoethanol, 40 μg / L hydrocortisone 21-hemisuccinate, and 40 μg / L progesterone. The culture medium also contained penicillin (90 U / mL) and streptomycin (0.09 mg / mL). The cells were grown at 37°C in a humidified atmosphere of 5% CO2 and 95% air.

[0151] Activity assay . The activity of caspase 3, caspase 8, and caspase 9 was measured using a colorimetric tetrapeptide cleavage assay kit according to the manufacturer's instructions (BioVision, Mountain View, Calif). A total of (661W / retina) protein was extracted according to a previously published protocol (Zacks et al., IOVS, 44(3): 1262-1267 (2003), incorporated herein by reference in its entirety). One hundred micrograms of total (661W / retina) protein was incubated for 60 minutes with a final concentration of 200 μM caspase 3 (DEVD-pNA), caspase 8 (IETD-pNA), or caspase 9 substrate (LEHD-pNA). The absorbance was measured at 405 nm in a microplate reader (Spectra-MAX 190, Molecular Devices, Sunnyvale, Calif). As a negative control, (661W / retina) protein was incubated with assay buffer without tetrapeptide. A second negative control was used in which the assay buffer alone was incubated with tetrapeptide. As positive controls, purified caspase 3, caspase 8, or caspase 9 were incubated with the tetrapeptide alone.

[0152] Previous experiments performed during the development of the present invention demonstrated that Fas signaling plays a key role in caspase 8 activation and photoreceptor apoptosis in vivo.

[0153] 661W cells were treated with FasL. Addition of FasL resulted in cell death. Caspase-8 activity measured in 661W cell lysates increased with increasing FasL concentration, reaching a peak at 500 ng / ml. 661W cells were treated with 500 ng / ml FasL, and activity levels were measured at various time points. Caspase-8 activity increased significantly after 48 hours of exposure of 661W cells to FasL. Across different runs, caspase-8 activity reliably increased by 20-30% in a dose-dependent manner.

[0154] The above-described assay system was used as an in vitro screening system to discover potential inhibitors of the Fas-induced caspase 8 activation pathway. When Met-12 (trihydrochloride) was tested in this 661W cell assay, it showed a dose-dependent reduction in FasL-induced caspase 8 activation, with a maximum at 10 μM, where caspase 8 activation was reduced to 0-25% of baseline, depending on the assay. This activity was highly dependent on the formulation in which the Met-12 peptide was delivered. The final formulation of Met-12 was diluted 1000-fold for use in this assay, and the top of the dose curve was typically 100 μM, with lesser dilutions used. Under these conditions, the greatest dose efficacy was observed with pure dimethyl sulfoxide solution, which delivers the Met-12 peptide as a clear, mobile liquid with an apparent pH well below 3.0. When aqueous-based formulations were tested, even in the absence of visual evidence of precipitation or aggregation before the material was added to the test wells, the formulations showed considerably less dose efficacy, with maximum inhibition not achieved until doses of 50-100 μM.

[0155] This strongly suggests that, regardless of the final physical form in the test wells, even in these cell assays, aggregation in the dosed solution results in much less druggable material than a true solution diluted to the exact same conditions, assuming the same inherent solubility potential. The most likely explanation to date is that the preformed aggregates in the non-solution state are kinetically and thermodynamically stable enough to not disaggregate into solution at the optimal rate during the assay, whereas the solution does not form aggregates upon dilution into the test wells, or more likely forms different aggregates that are more soluble. The primary reason for the different aggregates is that the peptide concentration is at least slightly lower when it is transferred from its pH- or cosolvent-enhanced soluble form to the 99% aqueous environment at pH 7.4. However, since effective mixing is not possible in the test wells, and is unlikely in the eye, and as discussed below, proton-accepting (low pH) and small molecule solvents will diffuse faster in water than hydrophobic and bulky peptides, it is highly likely that these peptides rapidly aggregated in the test wells or vitreous humor immediately after dosing. Thus, while solution dosing is clearly superior to suspension / gel dosing, there is no guarantee that significant amounts of the peptide will not be sequestered as insoluble aggregates and stochastically reduce the effective concentration of the drug upon administration.

[0156] Unexpectedly, if Figure 1 As shown, when Compound 1 trihydrochloride was tested in this assay as an unbuffered solution in DMSO, the IC 50 The assay demonstrated that it was 10-fold more potent than Met-12 and approximately 3-fold more potent as measured by the concentration that produced maximal inhibition of FasL-induced caspase 8 activation. 50The maximum inhibition was observed at 0.4 μM for Met-12 itself and 4 μM for Met-12 itself. However, above 3 μM, compound 1 showed a U-shaped curve and above 30 μM, it seemed almost inactive. In contrast, Met-12 dosed in the same manner reached its (slightly greater) maximum efficacy at 10 μM and then had only a slight rebound loss of efficacy beyond 100 μM. (See Figure 1 (Caspase 8 activity after 48 hours of treatment with human recombinant FasL after pretreatment with Met-12 and compound 1). 0% is the caspase 8 activity level of the untreated control. 100% is set to the caspase 8 activity of the control treated with FasL alone.

[0157] Since the mechanism of action of FasL involves the trimerization of the trivalent ligand at the Fas receptor, it is difficult to see how a monovalent Met-12 derivative could have a mixed agonist-antagonist dose profile, and the loss of potency is considered a solubility artifact of the assay. However, the unexpected increase in the dose potency of Compound 1 should allow for the administration of lower amounts of drug than would be required with Met-12 itself, which in turn reduces the solubility requirements of the peptide formulation for intravitreal injection.

[0158] Figure 2 The data shown in are consistent with the above explanation. It once again shows that Compound 1 trihydrochloride is slightly more dose-effective than Met-12 in DMSO (20 mg / mL), but it is again inactivated at higher concentrations. In contrast, when formulated at a concentration of 10 mg / mL in a transparent, filterable, potentially micellar solution of 2% polysorbate 20 and 2% PG at pH 4, the dose-effectiveness of Compound 1 trihydrochloride is significantly higher, but it is also more effective than Met-12 or Compound 1 in DMSO. In addition, there is little loss of maximum efficacy at the highest dose tested in this assay, 30 μM. This surprising result shows that the ability to formulate Compound 1 in a surfactant can lead to a significant improvement in drug efficacy.

[0159] Figure 3 A 20 mg / mL DMSO solution was compared to two polysorbate formulations (pH 4) of compound 1 trihydrochloride containing 1% and 3% PS-20 and 3% PG, all at 10 mg / mL. In this case, all three showed similar potency at lower concentrations, but 1% PS-20 showed only a slight loss of activity from its most effective concentration of 3 μM up to 30 μM, while 3% PS showed a continued increase in activity up to 30 μM, the highest dose tested.

[0160] exist Figure 4In Figure 1, an optimized formulation of compound 1 trihydrochloride (dark triangles) at a concentration of 2 mg / mL in 10 mM acetate buffer, 4.5% mannitol, and 0.4% PS-20 was compared to 20 mg / mL of compound 1 trihydrochloride in DMSO (light circles) in 661W cells. The trihydrochloride exhibited a typical U-shaped curve in DMSO, with a maximum effect at 3 μM and a considerable rebound at higher concentrations. Due to its low concentration, this formulation could only be tested up to 10 μM, but showed similar efficacy to the DMSO solution.

[0161] A possible explanation for the higher efficacy observed with micellar formulations is that peptide solubility is maintained for a longer time due to the surfactant's tendency to self-organize, which is only slowly affected by dilution and dispersion, in stark contrast to pH gradients or small molecule cosolvents. Consequently, the micelles and peptide are more widely dispersed into the aqueous medium before the peptide is released into the aqueous medium because the micelles only break apart slowly, whereas in cosolvents or highly acidic solutions, the solubilizing factors (hydrogen ions, small molecules) are very rapidly diluted into the aqueous medium before the peptide has any real chance to disperse outside the area directly where the injection is placed. This would result in less efficient dispersion of the peptide from a solution formulation and the formation of more inactive aggregates than if it had formed a micellar formulation.

[0162] Example 8: Intravitreal ocular pharmacokinetic study of compound 1 in rabbits

[0163] This study was conducted to determine the concentration of Compound 1 in the vitreous humor (VH) and retinal tissue following intravitreal injection in male Holland belt rabbits. Tissue concentrations were determined 24, 72, 168, and 240 hours after administration of a 50 μL bilateral intravitreal (IVT) dose.

[0164] The study design is summarized in Table 1 .

[0165] Table 1. Study design

[0166]

[0167] The test system includes the following:

[0168] Species / Strain / Gender: Male Dutch Belted Rabbits

[0169] Supplier: Covance Research Products, Inc.

[0170] Age range: 4 to 5 months

[0171] Weight at receipt (weight range): 1.51-1.85kg

[0172] Route of administration: Intravitreal (IVT) injection

[0173] Treatment duration: Single dose per eye

[0174] Formulation concentrations: 2.0, 1.0, and 0.5 mg / mL Compound 1 triacetate Dose volume: 50 μL per eye

[0175] An intravitreal (IVT) ocular dose of 50 μL was administered to the globe of each Dutch Black Strap rabbit eye.

[0176] At the corresponding time points, rabbits were euthanized by intravenous barbiturate overdose, and the eyes were enucleated and snap-frozen. Vitreous humor and retinas were collected from all animals and analyzed for compound 1 by LC-MS / MS.

[0177] calculate:

[0178] Percent coefficient of variation: Used as an estimate of precision.

[0179] Percent coefficient of variation (%CV) = (standard deviation / mean) * 100

[0180] Quadratic least squares analysis: Use with 1 / x 2 The standard curve was fitted using a quadratic equation with weights:

[0181] y=ax 2 +bx+c

[0182] Where: y = peak area ratio of calibration standard to internal standard

[0183] x = concentration of calibration standard

[0184] a=x 2 The coefficients of the quadratic equation

[0185] b = coefficient of the quadratic equation x

[0186] c = This constant is the y-intercept of the calibration curve

[0187] Analyte concentration from the quadratic equation: Calculate the analyte concentration using the calibration curve parameters calculated above, then solve for the value of x.

[0188] result

[0189] Retinal and VH concentrations are found in Tables 2 and 3 below and in Figure 1 and 2 The pharmacokinetic parameters were calculated as appropriate and are summarized in Tables 4 and 5 below.

[0190] Table 1. Compound 1 concentration in the retina of Dutch Black-Belt rabbits

[0191]

[0192]

[0193]

[0194]

[0195]

[0196] LLOQ = 20.0 ng / mL or 100 ng / g

[0197] Values < LLOQ were statistically determined using 0

[0198] ISD = Data not sufficient to determine

[0199] Underline: Exceeding the upper limit of the quantitative limit

[0200] As well as outliers, excluded data

[0201] Table 3. Compound 1 concentration in the vitreous humor of Dutch Black-Belt rabbits

[0202]

[0203]

[0204] [[ID=4 5]]

[0205]

[0206]

[0207] LLOQ = 50.0 ng / mL

[0208] *Assume density 1.00 g / mL

[0209] Underline: suspected outliers, data not included in the statistics

[0210] ISD = Data not sufficient to determine

[0211] Italic: Results exceed the upper limit of quantification, estimated data.

[0212] The results of this study in the retina and vitreous humor are shown in Figures 5 (A+B) and 6 (A+B), respectively. The retinal concentration of Compound 1 was variable over the time course of the study for all groups, with coefficients of variation (%CV) ranging from 52% to 156%. Retinal T was recorded 72 (3 days) or 168 (7 days) hours after administration of the three poloxamer formulations. max , but not dose-dependently, whereas polysorbate formulations had a max The retinal AUC was 24 hours after administration. 0-last Follow the C max Similar variable pattern. For compound 1, retinal T 1 / 2 Retinal data could only be calculated for 2 mg / mL poloxamer (168 hours) and 1 mg / mL polysorbate (199 hours).

[0213] Analysis of the vitreous humor of Compound 1 showed relatively consistent concentrations at each time point for the poloxamer formulations. When normalized by the weight of the collected VH, the theoretical total amount of Compound 1 was calculated. The total amount of Compound 1 injected into each rabbit eye was 100 μg (2 mg / mL * 50 μL) or 25 μg (0.5 mg / mL * 50 μL) for the poloxamer group and 50 μg (1 mg / mL * 50 μL) for the polysorbate group. The lowest mean concentration for all groups was at day 10. From 24 hours to 10 days after IVT administration, the average remaining Compound 1 in the VH of Group 1 (100 μg) ranged from 84.2 μg to 106 μg. From 24 hours to 10 days after IVT administration, the average remaining Compound 1 in the VH of Group 2 (25 μg) ranged from 19.5 μg to 23.0 μg. From 24 hours to 10 days after IVT administration, the average remaining compound 1 in the VH of Group 3 (25 μg) ranged from 26.0 μg to 20.4 μg. Following IVT administration, the average remaining compound 1 in the VH of Group 4 (50 μg) with polysorbate ranged from a high of 46.8 μg at 24 hours to 19.4 μg at 10 days. The polysorbate group was the only formulation to achieve a significant decrease in compound 1 concentration over time. However, significant amounts of intact drug were detected in all groups 10 days after administration.

[0214] Calculation of the pharmacokinetic parameters of compound 1 in VH indicated that T max , its C max The total amount administered to each group (Table 4) was closely matched. 0-lastRoughly dose proportional: Group 1 (2 mg / mL) had approximately four times the AUC compared to Groups 2 and 3 (both 0.5 mg / mL). The polysorbate group (1 mg / mL) was less than dose proportional relative to the poloxamer group, but this can be readily explained as it was the only group in which Compound 1 VH concentrations decreased significantly over the course of the study. T 1 / 2 The linear fit was good for 183 hours, while for the poloxamer group, the 1 / 2 >900 hours, the linear fit is not ideal.

[0215] Table 4. Pharmacokinetic parameters of compound 1 in vitreous humor

[0216]

[0217] *R2<0.7

[0218] Table 5. Pharmacokinetic parameters of compound 1 in the retina

[0219]

[0220] NC – Not Calculated

[0221] In summary, when administered IVT in either poloxamer or polysorbate formulations, Compound 1 showed no signs of irritation or tolerability issues over the course of the 10-day study. When administered IVT in a poloxamer formulation, Compound 1 concentrations in the retina or VH did not readily decrease over a period of up to 10 days, and possibly longer. When administered in a polysorbate formulation, Compound 1 showed a clear, albeit slow, decrease in concentration over the course of the 10-day study, suggesting that intravitreal pharmacokinetics can be controlled within certain parameters by careful selection of the nonionic surfactant used.

[0222] Example 9: Ocular Pharmacokinetic Study of Compound 1 in Rats

[0223] This study was conducted to determine the concentration of Compound 1 trihydrochloride in the vitreous humor and retinal tissue after intravitreal injection of Brown Norway rats. Tissue concentrations were determined 24 (Group 1) and 72 hours (Groups 1 and 2) after administration of a 5 μL bilateral intravitreal (IVT) dose.

[0224] The study design is summarized in Table 6 .

[0225] Table 6. Study design

[0226]

[0227] The test system includes the following:

[0228] Species / Strain / Gender: Norway Brown Rats

[0229] Supplier: Charles River, Inc.

[0230] Age Range: 1 to 2 months

[0231] Received Weight (Weight Range): 165.9–181.2 g

[0232] Administration Route: Intravitreal (IVT) injection

[0233] Treatment Duration: Single dose per eye

[0234] Formulation Concentration: 0.06 mg / mL ONL-1204 triacetate Dose Volume: 50 μL per eye

[0235] Procurement of ocular tissue

[0236] The rats were euthanized by intravenous barbiturate overdose, and the eyes were enucleated and snap-frozen. Vitreous humor (VH) and retina were collected from all animals, and Compound 1 was analyzed by LC-MS / MS.

[0237] result

[0238] Ocular Tissue Concentration of Compound 1 trihydrochloride in the retina and vitreous humor of brown Norway rats (unknowns).

[0239] Table 7. Concentration of Compound 1 in the retina of brown Norway rats

[0240]

[0241] LLOQ = 20.0 ng / mL or 100 ng / g

[0242] Values < LLOQ were statistically determined using 0

[0243] Footnote 1: Assumed to be 1.00 g / mL for rat retinal tissue

[0244] Table 8. Concentration of Compound 1 in the vitreous humor of brown Norway rats

[0245]

[0246] LLOQ = 50.0

[0247] ng / mL

[0248] *Assumed density of 1.00 g / mL

[0249] Tissue homogenization

[0250] Instructions for homogenization of vitreous humor (VH) unknowns

[0251] For each VH unknown or control blank: Weigh the VH into a homogenizer tube. Add 4 times the VH weight (mg) in ACN:water:1M HCl (70:20:10, v / v / v) (μL) to the homogenizer tube. Add zirconium oxide beads, 2.8 and 1.4 mm in size. Use All samples were homogenized under the following conditions: 5500 rpm, 3 × 30 s cycles with 20 s between cycles, and temperature between −10 °C and 0 °C.

[0252] Instructions for homogenization of retinal ocular tissue standards

[0253] For each retinal standard:

[0254] Weigh the blank retinal tissue into a homogenizer tube.

[0255] Add 0.5 times the tissue weight (mg) of retinal working calibration standard (μL) to the homogenization tube.

[0256] Add 3.5 times the tissue weight (mg) of ACN:water:1 M hydrochloric acid (70:20:10, v / v / v) (μL) to the homogenization tube.

[0257] Zirconia beads were added, with a size of 1.4 mm.

[0258] use All samples were homogenized under the following conditions: 5500 rpm, 3 × 30 s cycles with 20 s between cycles, and temperature between −10 °C and 0 °C.

[0259] Instructions for homogenization of retinal tissue blanks and unknowns

[0260] For each unknown retina or control blank:

[0261] Weigh the retinal tissue into a homogenizer tube.

[0262] Add 4 times the tissue weight (mg) of ACN:water:1 M hydrochloric acid (70:20:10, v / v / v) (μL) to the homogenization tube.

[0263] Zirconia beads were added, with a size of 1.4 mm.

[0264] use All samples were homogenized under the following conditions: 5500 rpm, 3 × 30 s cycles with 20 s between cycles, and temperature between −10 °C and 0 °C.

[0265] Preparation of standards, samples, and blanks

[0266] Preparation of calibration stocks and working standards

[0267] A stock calibration standard of ONL-1204 was prepared in dimethyl sulfoxide (DMSO) at a concentration of 500 μg / mL.

[0268] Working calibration standards for vitreous humor were prepared in the range of 500 ng / mL to 200,000 ng / mL of ONL-1204 by serially diluting the working stock solutions with ACN:w water:1 M hydrochloric acid (70:20:10, v / v / v).

[0269] Working calibration standards for retina were prepared in the range of 100 ng / mL to 200,000 ng / mL of ONL-1204 by serial dilution of the working stock solution with acetonitrile:water:1 M hydrochloric acid (70:20:10).

[0270] Preparation of standards, unknowns, blanks, and blanks with internal standards for vitreous humor analysis

[0271] In a polypropylene tube, add 10 μL (20 μL STD 11) of the working calibration standard or stock solution to 90 μL (80 μL STD 11) of the control blank vitreous humor. For the blank and the blank with internal standard, add 100 μL of the control blank bovine vitreous humor. Add 400 μL of ACN:water:1 M hydrochloric acid (70:20:10, v / v / v) to each standard or blank.

[0272] One hundred (100 μL) of each vitreous humor sample containing ACN:formic acid (1000:1, v / v) was then aliquoted. One hundred (100 μL) of DMSO:water:formic acid (50:40:10) was added to each vitreous humor sample. The samples were vortexed and then centrifuged at 14,000 rpm (4°C) for 10 minutes. To 50.0 μL of supernatant was added 100 μL of working internal standard (50,000 ng / mL APi1887 in water) (water was used as a blank without internal standard) and 150 μL of water. The samples were then vortexed and transferred to the autosampler plate for analysis.

[0273] Preparation of standards, unknowns, blanks, and blanks with internal standards for retinal analysis

[0274] In a polypropylene tube, 50 μL of brown Norway rat unknown homogenate, bovine control blank, or calibration standard bovine homogenate was added. Fifty (50) μL of DMSO:water:formic acid (50:40:10) was added to each sample. The samples were then vortexed and centrifuged at 14,000 rpm (4°C) for 10 minutes. Eighty (80) μL of each sample supernatant was then aliquoted into a 96-well autosampler plate. Forty (40) μL of working internal standard (5,000 ng / mL APi1887 in water) (water was used as a blank without internal standard) and 120 μL of water were added. The samples were then mixed with a multichannel pipette and analyzed.

[0275] calculate

[0276] Percent coefficient of variation: Used as an estimate of precision.

[0277] Percent coefficient of variation (%CV) = (standard deviation / mean) * 100

[0278] Quadratic least squares analysis: Use with 1 / x 2 The standard curve was fitted using a quadratic equation with weights:

[0279] y=ax 2 +bx+c

[0280] Where: y = peak area ratio of calibration standard to internal standard

[0281] x = concentration of calibration standard

[0282] a=x 2 The coefficients of the quadratic equation

[0283] b = coefficient of the quadratic equation x

[0284] c = This constant is the y-intercept of the calibration curve

[0285] Analyte concentration from the quadratic equation: Calculate the analyte concentration using the calibration curve parameters calculated above, then solve for the value of x.

[0286] analyze

[0287] The overall average results are Figure 7As shown in . For Group 1, both eyes from each animal were combined for a single analysis. In Group 2, samples were prepared from both eyes of four animals. The concentrations of the pooled retina in Group 2 were 462 and 1310 ng / g for AD and EH samples, respectively. Vitreous humor analysis of Compound 1 was performed using combined samples from each eye of each animal in Groups 1 and 2. When normalized by the weight of collected VH, the theoretical total amount of Compound 1 was calculated. The total amount of Compound 1 injected into each rat eye was 0.3 μg (0.06 mg / mL*5 μL). Due to different methods, 72 hours of data were collected and no standard error was calculated. The dark bars represent the amount of Compound 1 triacetate in the vitreous humor of each eye, where the bar at t=0 represents the expected dose of 300 ng, and the light bars represent the concentration of Compound 1 in the retina, expressed in ng / g. Within the first 24 hours, about half of the drug cleared the VH, but the final half-life was obviously around a few days. Meanwhile, retinal concentrations exceeded 1 μg / g at 24 hours, but were only about 40% of that at 72 hours, suggesting that the rat retina was exposed to the drug in easily detectable amounts for at least a week.

[0288] Retinal concentrations of Compound 1 ranged from 146 to 3670 ng / g in Group 1 24-hour samples and from 409 to 804 ng / g in Group 1 72-hour samples. The average remaining Compound 1 in the VH of Group 1 was 0.157 and 0.0994 μg / sample at the 24-hour and 72-hour time points, respectively. At 72 hours, the average remaining Compound 1 in the VH of Group 2 samples was 0.127 μg / sample. The data indicate that a significant amount of intact drug was still present 72 hours after administration.

[0289] Example 10: In vivo efficacy of compound 1

[0290] Briefly, rodents were anesthetized with a 50:50 mixture of ketamine (100 mg / mL) and xylazine (20 mg / mL), and the pupils were dilated with topical phenylephrine (2.5%) and tropicamide (1%). A scleral incision was made 2 mm posterior to the limbus using a No. 20 microvitreoretinal blade (Walcott Scientific, Marmora, NJ), taking care to avoid lens damage.

[0291] Under direct visualization through an operating microscope, a subretinal syringe (Glaser, 32-gauge tip; BD Ophthalmic Systems, Sarasota, FL) was introduced into the vitreous cavity through a sclerotomy and then into the subretinal space through a peripheral retinotomy. Sodium hyaluronate (10 mg / mL) was slowly injected to detach the neurosensory retina from the underlying retinal pigment epithelium.

[0292] In all experiments, approximately one-third to one-half of the superonasal neurosensory retina was detached. In all animals, detachments were generated in the same location to allow direct comparison of retinal cell counts. Detachments were generated in the left eye, with the right eye serving as a control.

[0293] In some eyes, wild-type Met-12 (HHIYLGAVNYIY, 5 μg in DMSO) was administered as its trihydrochloride salt as a positive control, while in other eyes, compound 1 (0.5, 1.0, 5.0, or 10 μg in DMSO) was injected as its trihydrochloride salt or vehicle (dimethyl sulfoxide [DMSO]) into the subretinal space of the detachment area immediately after the detachment was established using a Hamilton syringe (Hamilton Company, Reno, NV) in a volume of 5 μL.

[0294] Three days later, the rats were euthanized and the retinas were dissected, fixed, sliced, and stained for TUNEL assay. The area of ​​the detached retina was counted for the number of apoptotic cells. Each experiment involved 4 fields of view in each of 4 sections obtained from 5 or 6 retinas of each dose group. The results are shown in Figure 8 middle.

[0295] like Figure 8 As shown, control attached retinas showed no apoptotic cells, while DMSO control retinas had approximately 4% of cells staining for TUNEL, while the positive control Met-12 (5 μg) had slightly over 1% positive for TUNEL. Compound 1 trihydrochloride treatment resulted in 0.58% at 0.5 μg, 1.14% at 1 μg, 0.82% at 5 μg, and 0.9% at 10 μg. Based on considerable experience with the model, the results for Compound 1 are expected to be roughly equivalent to each other and to the Met-12 5 μg positive control. Therefore, unexpectedly, Compound 1 trihydrochloride is more effective than Met-12 trihydrochloride in this in vivo model.

[0296] Example 11: Efficacy Study

[0297] Using the same rodent retinal detachment model as in Example 10, a highly effective 5 μg dose of Compound 1 in DMSO was compared to the same dose and a 1 μg dose of Compound 1 in two different formulations.

[0298] The first formulation was a 1.0 or 0.2 mg / mL solution of compound 1 trihydrochloride in 3% propylene glycol and 3% PS-20, and the second formulation was the same concentration of compound 1 in 2% propylene glycol and 2% poloxamer 407, also at a pH of 4.0. Figure 9 Shown in.

[0299] Attached control retinas showed no apoptotic cells, whereas untreated detachments showed approximately 6.5% apoptotic cells measured at this time point.

[0300] Compound 1 reduced this to 2.5% in DMSO, and 5 μg of PG / PS-20 had similar potency, reducing apoptotic cells to 2.9%. However, the PG / PX formulation was quite good at reducing apoptotic cells to 0.3% at 5 μg. At 1 μg, the PG / PX formulation reduced apoptotic cells to 0.4%, but the 1 μg dose of PG / PS-20 produced a greater reduction, to 0.01%. The data suggest that not only can micelle formation play a role, but that compound 1 trihydrochloride can be more effective in it than when formulated in DMSO.

[0301] Example 12: Efficacy Study

[0302] The same rodent retinal detachment model as in Example 10 was used, using Compound 1 trihydrochloride in DMSO (1 μg) as a positive control. The negative control was the test vehicle being evaluated (0.4% poloxamer, 4.5% mannitol, 10 mM acetic acid, pH 4.5). Compound 1 triacetate in DMSO (1 μg) was compared to the same dose of the trihydrochloride.

[0303] The attached retina (bar 4) had no apoptotic cells, while the vehicle-treated detached retina (bar 1) showed 4.2% apoptotic cells, which indicates no activity as it is within the historical range of untreated retinal detachments (see Example 10). Compound 1 trihydrochloride (DMSO) produced 2.4% apoptotic cells, while triacetate (DMSO) produced only 1.2%, demonstrating that switching from the hydrochloride to the acetate salt has no negative effect and may have a positive effect on efficacy.

[0304] All publications and patents mentioned in this application and / or listed below are incorporated herein by reference. Without departing from the scope and spirit of the present invention, various modifications and variations of the method and composition of the present invention will be apparent to those skilled in the art. Although the present invention has been described in conjunction with specific preferred embodiments, it should be understood that the claimed invention should not be unduly limited to these specific embodiments. In fact, the various modifications described for implementing the mode of the present invention are apparent to those skilled in the relevant art and are intended to be within the scope of the following claims.

[0305] Although the present invention has been described in considerable detail with reference to certain embodiments thereof, other embodiments are possible without departing from the present invention. Therefore, the spirit and scope of the appended claims should not be limited to the description of the preferred embodiments contained herein. All embodiments that fall within the meaning of the claims, whether literally or by equivalents, are intended to be encompassed therein.

[0306] Furthermore, the advantages described above are not necessarily the only advantages of the present invention, and it is not necessarily expected that all of the described advantages will be achieved by every embodiment of the present invention. Sequence Listing <110> ONL THERAPEUTICS LIMITED. University of Michigan Board of Regents <120> Peptide compositions and methods of use <130> 15428-9 <150> US 62 / 155,711 <151> 2015-05-01 <160> 2 <170> PatentIn version 3.5 <210> 1 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Artificial peptide; C-terminal amide of Met-12 <400> 1 His His Ile Tyr Leu Gly Ala Val Asn Tyr Ile Tyr 1 5 10 <210> 2 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Artificial peptide; small molecule peptide inhibitor, Met-12 <400> 2 His His Ile Tyr Leu Gly Ala Val Asn Tyr Ile Tyr 1 5 10

Claims

1. A compound comprising formula 1: Use of a pharmaceutical composition of a compound or a pharmaceutically acceptable salt thereof for the preparation of a medicament for treating Fas-mediated or TRAIL-mediated photoreceptor cell death in the eye of a subject, wherein the pharmaceutical composition comprises 0.1 mg to 2.0 mg of the compound or a pharmaceutically acceptable salt thereof.

2. A compound comprising formula 1: or a pharmaceutically acceptable salt thereof for preparing a medicament for treating Fas-mediated or TRAIL-mediated retinal pigment epithelial apoptosis or necrosis in the eye of a subject, wherein the pharmaceutical composition comprises 0.1 mg to 2.0 mg of the compound or a pharmaceutically acceptable salt thereof.

3. Use of a pharmaceutical composition according to claim 1 or 2, wherein the pharmaceutical composition is formulated for ocular delivery.

4. The use of the pharmaceutical composition according to claim 3, wherein the pharmaceutical composition is administered to the subject intraocularly, intravitreally or periocularly.

5. Use of a pharmaceutical composition according to claim 4, wherein the pharmaceutical composition comprises at least one nonionic surfactant.

6. Use of the pharmaceutical composition according to claim 5, wherein the at least one nonionic surfactant is selected from polysorbate 80, polysorbate 20, poloxamer 407 and tyloxapol.

7. Use of a pharmaceutical composition according to claim 6, wherein the at least one non-ionic surfactant comprises 0.05% w / w to 10% w / w of the composition.

8. Use of a pharmaceutical composition according to claim 7, wherein the at least one non-ionic surfactant comprises 0.25% w / w to 3% w / w of the composition.

9. The use of the pharmaceutical composition according to claim 5, wherein the pharmaceutical composition further comprises an organic cosolvent.

10. The use of the pharmaceutical composition according to claim 9, wherein the organic co-solvent comprises 1% w / w to 20% w / w of the composition.

11. The use of the pharmaceutical composition according to claim 10, wherein the organic co-solvent accounts for 1% w / w to 5% w / w of the composition.

Images