Compositions comprising ped-derived oligopeptides (PDSP) and

Abstract

An aqueous formulation comprising: a pigment epithelium-derived factor (PEDF)-derived oligopeptide (PDSP); a mixture of polyvinylpyrrolidone (PVP) and polyvinyl alcohol (PVA) in a ratio of about 8 / 2 to 2 / 8; and a tension agent, preferably a nonionic tension agent. The PDSP is preferably 14-39 amino acids in length and has a sequence of one of SEQ ID NO: 1, 2, 3, 5, 6, 8 or 9. And the nonionic tension agent is glycerol, cane sugar, mannitol or sorbitol. And the concentration of the PDSP is 0.01%-1% w / v.

Description

BACKGROUND OF THE INVENTION FIELD OF THE INVENTION

[0001] The present invention relates to compositions of PEDF-derived short peptides, particularly formulations of such peptides and their uses. BACKGROUND

[0002] Human pigment epithelium-derived factor (PEDF) is a secreted protein composed of 418 amino acids with a molecular weight of approximately 50 kDa. PEDF is a multifunctional protein with a variety of biological functions (see U.S. Patent Application Publication No. 2010 / 0047212). It has been found that different peptide segments of human PEDF are responsible for different functions. For example, a 34-mer fragment (amino acids 44 - 77 of PEDF) has been shown to have anti-angiogenic activity, while a 44-peptide fragment (amino acids 78 - 121 of PEDF) has neurotrophic properties.

[0003] Human PEDF-derived short peptides (PDSPs) have been found to be promising therapeutic agents for treating or preventing a variety of diseases or disorders. For example, PDSPs have been shown to be effective in promoting muscle regeneration or arteriogenesis (U.S. Patent No. 9,884,012), treating hair loss and / or hair depigmentation (U.S. Patent No. 9,938,328), treating osteoarthritis (U.S. Patent No. 9,777,048), preventing or improving skin aging (U.S. Patent No. 9,815,878), treating liver cirrhosis (U.S. Patent No. 8,507,446), or treating various eye diseases or disorders such as retinal degeneration, meibomian gland diseases, and dry eye. The corresponding murine PEDF-derived short peptides (moPDSPs) have also been found to have the same therapeutic effects. However, the formulations of these peptides have been found to lack long-term stability. Therefore, it is necessary to develop better formulations for this promising biopharmaceutical product. SUMMARY OF THE INVENTION

[0004] Embodiments of the present invention relate to formulations of PEDF-derived short peptides (PDSPs), including SEQ ID NO:1 (39-mer), SEQ ID NO:2 (34-mer), SEQ ID NO:3 (29-mer), SEQ ID NO:5 (24-mer), SEQ ID NO:6 (20-mer), SEQ ID NO:8 (mo29-mer), and SEQ ID NO:9 (mo20-mer), where the mo29-mer and mo20-mer are murine PDSPs corresponding to the human 29-mer and 20-mer, respectively. Our recent studies have found that the core peptide retaining the biological activity of PDSP is located in the first 14 amino acids of SEQ ID NO:3. Although embodiments of the present invention can use PDSPs of any length, preferred embodiments contain PDSPs that are 14 - 39 amino acids in length.

[0005] One aspect of the present invention relates to an aqueous preparation comprising PDSP, polyvinylpyrrolidone (PVP), polyvinyl alcohol (PVA), and a tensioactive agent. The tensioactive agent is preferably a nonionic tensioactive agent. The nonionic tensioactive agent can be any tensioactive agent suitable for pharmaceutical preparations, such as glycerol, sucrose, mannitol, or sorbitol. PDSP can be 14 - 39 amino acids in length, its N-terminus can be protected by an amide, and / or its C-terminus can be an ester or an amide. PDSP preferably has a sequence of one of SEQ ID NO:1, 2, 3, 5, 6, 8, or 9. The ratio of PVP / PVA is preferably between 9 / 1 and 1 / 9, such as 8 / 2, 6 / 4, 4 / 6, or 2 / 8. The aqueous preparation further comprises a buffer, preferably a histidine buffer.

[0006] According to some embodiments of the present invention, the pH value of the aqueous preparation is about 5 - 9, preferably about 6 - 8, more preferably about 6.5 - 7.5. The nonionic tensioactive agent is preferably sorbitol, and its concentration is 10 mM - 500 mM, preferably 50 - 250 mM, more preferably 100 - 150 mM. The concentration of PDSP can be 0.01% - 10% w / v, preferably 0.01% - 1% w / v, more preferably about 0.02% - 0.2% w / v.

[0007] Other aspects of the present invention will be apparent from the following description and the drawings. Brief Description of the Drawings

[0008] Figure 1Shows a schematic diagram of the test protocol for evaluating the stability of various formulations of PDSP solution. According to the research design, PVP and PVA (hereinafter referred to as "PVP / PVA") are mixed in different ratios to form different PDSP solutions. First part: Select different PVP / PVA ratios, adjust the pH value of the PDSP solution with 1N HCl or 2N NaOH, filter through a 0.2μm needle filter, and then place each solution in a 20ml glass bottle with a screw cap. The filtered PDSP solution is stirred at 1,100 RPM at room temperature. Collect 80μl of the PDSP solution at different time points, and determine the size distribution of small particles in different formulation solutions by dynamic light scattering (DLS). Continue to stir the PDSP solution, and further study the precipitation by centrifugation at 13,000 rpm at the 48-hour time point. Second part: Particle analysis, filter the PDSP solution with different PVP / PVA ratio formulations through a 0.2μm syringe filter, and then place each solution in a 50ml glass bottle with a screw cap. Stir the filtered PDSP solution at 390 RPM at room temperature for 24 hours. Then, check for particulate matter in the formulation solution according to the United States Pharmacopeia (USP) <789> test. If the average number of particles present in the test unit does not exceed the appropriate value listed in Table 1, the solution passes the USP <789> test. Table 1: Particle number limit for USP <789> test

[0009] Figure 2 Shows the stability test results of PDSP formulations prepared at different PVP / PVA ratios (10 / 0, 8 / 2, 6 / 4, 4 / 6, 2 / 8, and 0 / 10 respectively) under continuous stirring conditions. These solutions were stirred at 1,100 RPM at room temperature after filtration and centrifuged after 48 hours. The precipitation of different PVP / PVA ratio formulations containing 0.03% PDSP is shown as a peak to the right of the main peak in the graph.

[0010] Figure 3 Shows the stability test results of PDSP formulations prepared at different PVP / PVA ratios under continuous stirring conditions. These solutions (with or without 0.03% PDSP) were continuously stirred at 1,100 RPM at room temperature. The particle size distribution was recorded by DLS at 0, 3, 5, 8, 24, and 48 hours. Finally, particle formation was evaluated by centrifugation at 48 hours. Detailed description of the invention

[0011] Embodiments of the present invention relate to PEDF-derived short peptide (PDSP) formulations with enhanced stability. A variety of human PDSPs have been found to be promising therapeutic agents for treating or preventing a variety of diseases or disorders, including muscle regeneration or angiogenesis, hair loss and / or hair depigmentation, osteoarthritis, skin aging, liver cirrhosis, or eye diseases or disorders. Examples of such PDSPs may include the peptides shown in Table 2. Table 2: Examples of PEDF-derived short peptides (PDSPs)

[0012] According to an embodiment of the present invention, the PDSP can be SEQ ID NO: 1, 2, 3, 5, 6, 8, or 9. In addition, the N-terminus of these peptides can be selectively protected by acylation (e.g., acetylation or propionylation), and the C-terminus can be selectively protected by amide.

[0013] These PDSPs have been prepared in citrate buffer and have been found to have therapeutic effects in various preclinical studies. However, formulations of these short peptides (e.g., BRM421, 29-mer PDSP (SEQ ID NO: 3) in 10 mM citrate buffer containing 0.85% w / v NaCl, pH 6.0) have been found to lack long-term (several months) stability.

[0014] Many factors, including chemical stresses (e.g., oxidation, hydrolysis, etc.) and physical stresses (e.g., temperature, light, and agitation), can affect the quality and stability of biopharmaceutical products, especially during long-term storage. To study the stability of PDSP in different formulations, accelerated stability tests were conducted. Specifically, various formulations were tested under stress conditions, especially under shear stress, to determine the optimal formulation.

[0015] Polyvinylpyrrolidone (PVP) is a water-soluble, inert, non-toxic, pH-stable biocompatible polymer that helps dissolve hydrophilic and lipophilic drugs. These advantages make PVP a versatile excipient in pharmaceutical formulation development. Polyvinyl alcohol (PVA) is a water-soluble polymer commonly used to increase the viscosity of pharmaceutical formulations and as a lubricant and protective agent in ophthalmic formulations. The use of the PVP / PVA combination in ophthalmic formulations has been found to have a soothing effect. We studied the ability of this combination to provide long-term stability to PDSP formulations. Embodiments of the present invention show that the stability of PDSP solutions can be enhanced at certain PVP / PVA ratios.

[0016] The following describes specific examples to illustrate embodiments of the present invention. However, those skilled in the art should understand that these specific examples are for illustration only, and other modifications and variations are possible without departing from the scope of the present invention. For example, although the following examples use BRM421 (29-mer PDSP; SEQ ID NO: 3) for illustration, other PDSPs can also be used. 1. Preparation of Formulations with Different PVP / PVA Ratios

[0017] PVP / PVA formulations can be used at any suitable concentration, such as 0.5 - 10% w / v, preferably 1 - 5% w / v, more preferably 2 - 4% w / v. PVP and PVA can be easily obtained from commercial sources (such as Sigma-Aldrich). The number-average molecular weight of PVP applicable to the embodiments of the present invention can be in the range of 10 - 400 kDa, preferably 10 - 100 kDa, more preferably 20 - 60 kDa. The weight-average molecular weight range of PVA applicable to the embodiments of the present invention can be in the range of 10 - 100 kDa, preferably 10 - 50 kDa, more preferably 10 - 30 kDa.

[0018] As an example, the preparation of a 2% w / v PVP / PVA formulation is described below. Those of ordinary skill in the art should understand that these examples are for illustration only, and other percentages (such as 3%, 4%, etc.) and / or PVP / PVA ratios can be prepared similarly. First, solutions of 2% PVP (number-average molecular weight of about 60,000) and 2% PVA (weight-average molecular weight of about 13,000) are separately prepared in distilled water. Then, they are physically mixed in ratios of 10 / 0, 8 / 2, 6 / 4, 4 / 6, 2 / 8, and 0 / 10 for study. For example, for an 8-milliliter solution with a 6 / 4 PVP / PVA ratio, the following material volumes were prepared: 8 milliliters of 2% PVP / PVA ratio = 6 / 4: 4.8 milliliters of 2% PVP + 3.2 milliliters of 2% PVA

[0019] Various tonicity agents have been used in ophthalmic formulations, such as citrate (MW 192.12), NaCl (MW 58.44), sorbitol (MW 182.17), and histidine (MW 155.15). The above tonicity agents can be added to the PVP / PVA solution to prepare a series of ophthalmic formulations for evaluating their ability to ensure long-term stability. The following are some examples: 1. Different ratios of 2% PVP / PVA solution with citrate buffer (55 mM citrate and 45 mM NaCl, pH 7.5); 2. Different ratios of 2% PVP / PVA solution with citrate buffer (55 mM citrate and 137 mM sorbitol, pH 7.5); 3. 2% PVP / PVA solutions and histidine buffer (125 mM histidine and 137 mM sorbitol, pH 7.6) at different ratios. 2. Preparation of PDSP in different formulations

[0020] The main PDSPs used in these examples include BRM421 (29-mer) (SEQ ID NO: 3, MW 3243.6; peptide content: 88.6%), 39-mer (SEQ ID NO: 1; MW 4211.71; peptide content: 96.5%), and 20-mer (SEQ ID NO: 6, MW 2376.63; peptide content: 95.2%). PDSP can be used at any suitable concentration (e.g., 0.01% - 5% w / v, preferably 0.01% - 1% w / v, more preferably 0.01% - 0.1% w / v). In the following examples, the PDSP concentration is 0.03% w / v (about 0.1 mM). For example, the calculation formula for BRM421 (29mer) in 30 mL of solution is as follows: 0.03% BRM421 = 0.03 g / 100 ml = 0.0003 g / ml (about 0.1 mM) 0.0003 g ÷ 88.6% peptide content = 0.0003386 g 0.0003386 × 30 = 0.010158 g For example, 30 ml of 2% PVP / PVA ratio (8 / 2) solution plus citrate buffer + 0.010158 g of BRM421 = 30 ml, 0.03% BRM421

[0021] After the PDSP is completely dissolved in the solution, the pH value of the formulation is measured, and then adjusted to 7.5 or 7.6 according to the research design. Before use in the study, the PDSP formulation solution is filtered through an injection filter (e.g., 0.2 μm filter). 3. Evaluation of the shear resistance ability of PDSP in different formulations

[0022] We noted that early PDSP citrate buffer formulations were unstable during long-term storage. To test the effect of different PVP / PVA formulations on stability, accelerated change tests were performed on various PDSP formulations under stress conditions (e.g., shear stress).

[0023] For the first part of the study: Different PVP / PVA ratios were selected and 29mer PDSP solutions prepared in different buffers and excipients (as shown in Table 3) were stirred at 1,100 RPM at room temperature. Aliquots of 80 μl of the PDSP solution were collected at different time points into 1.5 ml Eppendorf tubes to determine the size distribution curves of the small particles in the different formulated solutions using dynamic light scattering (DLS). The PDSP solution was continuously stirred and precipitation was further investigated at the 48-hour time point after the start of stirring by centrifuging 300 μl of the PDSP solution at 13,000 rpm.

[0024] For the second part: Particle analysis, PDSP solutions of different PVP / PVA ratio formulations were stirred at 390 RPM at room temperature for 24 hours. Then, the formulated solutions were evaluated according to the USP <789> protocol. If the average number of particles present in the unit does not exceed the appropriate value, the formulation passes the USP <789> test. The experimental procedures are as Figure 1 shown. Table 3. List of formulations tested in this study. Results 1. Shear resistance ability of 29-mer PDSP prepared in solutions of different PVP / PVA ratios (10 / 0, 8 / 2, 6 / 4, 4 / 6, 2 / 8, and 0 / 10 respectively), the solution containing 55 mM citrate and 137 mM sorbitol, pH 7.5

[0025] The stability of PDSP under stirring conditions in different PVP / PVA ratio formulations was investigated by dynamic light scattering (DLS) and centrifugation analysis to elucidate the shear resistance ability of these formulations. The results showed that the formulation with a PVP / PVA ratio of 8 / 2 performed best in terms of shear resistance. In contrast, all other PVP / PVA ratios were less effective in protecting the PDSP formulation from shear forces, as evidenced by the formation of large amounts of precipitate / aggregates.

[0026] In aqueous solution, precipitation is caused by the process of converting dissolved PDSP from the formulated solution into insoluble solids under stress conditions. At 48 hours after the start of stirring, precipitation analysis based on high-speed centrifugation of particle formation showed that no precipitation was observed for the formulation with a PVP / PVA ratio = 8 / 2. In contrast, the other formulations showed significant precipitation ( Figure 3 ). These data indicate that a 2% w / v PVP / PVA formulation with a ratio of approximately 8 / 2 is more suitable for PDSP stability. However, as described in the later part, different PVP / PVA ratios may be more preferred under different concentrations or different buffers and / or tonicity agents. 2. Particle analysis of 29-mer PDSP prepared with a PVP / PVA ratio of 8 / 2 under stirring conditions and different excipients

[0027] Ophthalmic solutions should be free of visually observable particles. USP <789> describes the test for counting particles in a specific size range. The particle matter of various PDSP preparations with different excipients and a PVP / PVA ratio of 8 / 2 was evaluated according to the USP <789> test. The PDSP preparation with a PVP / PVA ratio of 8 / 2, 55 mM citrate, 137 mM sorbitol, and pH 7.5 was the initial preparation. However, due to the large amount of pH adjustment to pH 7.5, this preparation was not conducive to PDSP preparation development. Therefore, other PDSP preparations with a PVP / PVA ratio of 8 / 2 and other tonicity regulators were studied. Particle analysis of 29-mer PDSP prepared with different excipients based on a PVP / PVA ratio of 8 / 2 under stirring conditions.

[0028] According to the particle test in ophthalmic solutions of USP <789>, the mixing effects of various PDSP preparations with a PVP / PVA ratio of 8 / 2 and other tonicity agents were evaluated under stirring conditions. The results are shown in Table 4. Since the results of the 45 mM NaCl preparation were not reproducible, the formulation with a PVP / PVA ratio of 8 / 2, 125 mM histidine, 137 mM sorbitol, and pH 7.6 was selected as the optimal formulation for the PDSP ophthalmic solution. Table 4. Particle analysis of 29-mer PDPS prepared with different formulations under stirring conditions Note 1. These three groups of studies were conducted twice to confirm the results, and the same results were obtained for the two formulations. 2. Out of range 3. Shear stress resistance of different PEDF-derived short peptides prepared in 2% and 4% PVP / PVA solutions with different ratios

[0029] The experiments on the PVP / PVA-based formulations described above were conducted using the 29-mer synthetic peptide SEQ ID NO:3. To investigate whether other PEDF-derived short peptides could also withstand agitation forces through PVP / PVA formulations, other PDSP short peptides, such as the 39-mer (SEQ ID NO:1) and the 20-mer (SEQ ID NO:6), were formulated at different PVP / PVA ratios as shown in Table 3. Part II of the study was conducted using these other PDSPs under the same shear force challenge. The results for the 39-mer and the 20-mer are shown in Table 5. These results together indicate that, in addition to the PVP / PVA ratio = 8 / 2, other PVP / PVA ratio formulations (PVP / PVA-based solutions containing 125 mM histidine and 137 mM sorbitol, pH 7.6) can also provide protection against shear stress, even for longer peptides (such as the 39-mer), and that PVP / PVA formulations with different PDSPs (such as SEQ ID NO:1, 2, 3, 5, 6, 8, and 9) also have enhanced stability.

[0030] Compared to the PDSP in citrate buffer, the PVP / PVA formulation was significantly more stable under the same stress conditions, demonstrating the superiority of the PVP / PVA formulation. Table 5. Particle size analysis of different PDSPs formulated with different PVP / PVA ratios under agitation conditions * Out of range

[0031] The above examples clearly show that, compared to the original formulation in citrate-based buffer, the newly formulated PDSP in different PVP / PVA-based solutions together with histidine or other tonicity agents can provide significantly improved stability.

[0032] Further experiments examined the effect of different PVP / PVA concentrations on the stability of PDSP formation. As shown in Table 6 below, at 4% PVP / PVA, PVP / PVA ratios of 4:6 and 2:8 produced better results. These results indicate that, according to the embodiments of the present invention, different concentrations and different ratios of PVP / PVA can be selected to suit different applications. Table 6: Different ratios of 4% PVP / PVA formulations #PDSP 0.1 mM in 350 mM nicotinamide, 20 mM histidine (without PVP / PVA) as a control * Out of range

[0033] Further experiments were conducted to examine the effect of different concentrations of PDSP (e.g., 29-mer) in PVP / PVA (4:6, 125 mM histidine, 130 mM sorbitol) on the stability of the PDSP formulation. As shown in Table 7 below, in PVP / PVA (4:6, 125 mM histidine, 130 mM sorbitol), PDSP at concentrations of 0.1–0.4 mM all had satisfactory stability (meeting the requirements of USP <789>) after 24 hours of forced aggregation (stirring at 390 rpm) at room temperature. These results indicate that the PVP / PVA formulation of the present invention can improve the stability of PDSP solutions at various concentrations for therapeutic applications. Table 7: Different Concentrations of PDSP in 0.2 mM PVP / 0.66 mM PVA (4:6), 125 mM Histidine, 130 mM Sorbitol #PDSP 0.1 mM in 350 mM nicotinamide, 20 mM histidine (without PVP / PVA) control group; · PVP K30: Sigma [catalog 81420] average molecular weight 40,000; · PVA Sigma [catalog 348406] average molecular weight 13,000–23,000, 98% hydrolyzed.

[0034] The above results show that PVP / PVA in various ratios (e.g., 2 / 8–8 / 2) and various concentrations (e.g., 1%, 2%, 3%, 4%, 5% w / v, etc.) can be used according to the embodiments of the present invention to improve the stability of the PDSP formulation. These PVP / PVA formulations can be used with PDSP at various concentrations (e.g., 0.01 mM–10 mM, preferably 0.1–1 mM, more preferably 0.1-0.5 mM). Compared with the citrate formulation of PDSP, the PVP / PVA formulation of PDSP is significantly more stable (see Table 5).

[0035] The embodiments of the present invention have been illustrated by only a limited number of examples. Those skilled in the art should understand that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention, as other modifications and variations can be made without departing from the scope of the present invention. Therefore, the scope of the present invention should be defined by the appended claims.

Claims

1. An aqueous preparation comprising: A pigment epithelium-derived factor (PEDF)-derived short peptide (PDSP); A mixture of polyvinylpyrrolidone (PVP) and polyvinyl alcohol (PVA); and A tensioactive agent.

2. The aqueous preparation according to claim 1, wherein the PDSP has a length of 14 - 39 amino acids.

3. The aqueous preparation according to claim 1, wherein the PDSP has the sequence of SEQ ID NO: 1, 2, 3, 5, 6, 8 or 9.

4. The aqueous preparation according to claim 1, wherein the PVP / PVA ratio of the mixture of PVP and PVA is 8 / 2, 6 / 4, 4 / 6 or 2 / 8.

5. The aqueous preparation according to claim 1, wherein the pH value of the aqueous preparation is 6.0 - 8.

0.

6. The aqueous preparation according to claim 1, wherein the tensioactive agent is a non-ionic tensioactive agent.

7. The aqueous preparation according to claim 6, wherein the non-ionic tensioactive agent is glycerol, sucrose, mannitol or sorbitol.

8. The aqueous preparation according to claim 6, wherein the non-ionic tensioactive agent is sorbitol.

9. The aqueous preparation according to claim 1, further comprising histidine.

10. The aqueous preparation according to claim 1, wherein the concentration of the PDSP is 0.01% - 1% w / v.

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