Compositions containing PEDF-derived short peptides (PDSPs) and uses thereof

Abstract

The aqueous formulation contains a PEDF-derived short-chain peptide (PDSP), a PVP / PVA mixture in a ratio of approximately 8 / 2 to 2 / 8, and preferably a non-ionic tonicity agent. The PDSP is preferably 14 to 39 amino acids in length and has the sequence of SEQ ID NO: 1, 2, 3, 5, 6, 8, or 9. The non-ionic tonicity agent is glycerin, sucrose, mannitol, or sorbitol. The concentration of the PDSP is 0.01% to 1% w / v.

Description

[Technical Field]

[0001] The present invention relates to compositions of short peptides derived from PEDF, in particular to the formulation of such peptides and their uses. [Background technology]

[0002] Human pigment epithelium-derived factor (PEDF) is a secreted protein consisting of 418 amino acids and has a molecular weight of approximately 50 kDa. PEDF is a multifunctional protein with many biological functions (see U.S. Patent Application Publication No. 2010 / 0047212). Different peptide regions of human PEDF have been shown to have distinct functions. For example, a 34-mer fragment (residues 44-77 of PEDF) has been shown to have anti-angiogenic properties, and a 44-mer fragment (residues 78-121 of PEDF) has been shown to have neurotrophic properties.

[0003] Human PEDF-derived short peptides (PDSPs) have been shown 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 angiogenesis (U.S. Patent No. 9,884,012), treating alopecia 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), and treating various ocular diseases or conditions (e.g., retinitis pigmentosa, meibomian gland dysfunction, dry eye). The corresponding mouse PEDF-derived short peptides (moPDSPs) have also been shown to have the same therapeutic effects. However, formulations of these peptides have proven to lack long-term stability. Therefore, better formulations are needed for this promising biopharmaceutical. 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 mouse PDSPs corresponding to the human 29-mer and 20-mer, respectively. Our recent studies have revealed that the core peptide that retains the biological activity of PDSP is located within the first 14 residues of SEQ ID NO:3. While embodiments of the present invention can utilize PDSPs of any length, preferred embodiments of the present invention include PDSPs between 14 and 39 amino acids in length.

[0005] One aspect of the present invention relates to an aqueous formulation comprising PDSP, polyvinylpyrrolidone (PVP), polyvinyl alcohol (PVA), and an isotonicity agent. The isotonicity agent is preferably a non-ionic isotonicity agent. The non-ionic isotonicity agent may be any isotonicity agent suitable for pharmaceutical formulations, such as glycerin, sucrose, mannitol, or sorbitol. The PDSP may be 14 to 39 amino acids in length, its N-terminus may be protected as an amide, and / or its C-terminus may be an ester or amide. The PDSP preferably has the sequence of SEQ ID NO: 1, 2, 3, 5, 6, 8, or 9. The PVP / PVA ratio is preferably in the range of 9 / 1 to 1 / 9, e.g., 8 / 2, 6 / 4, 4 / 6, or 2 / 8. The aqueous formulation further comprises a buffer, preferably a histidine buffer.

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

[0007] Other aspects of the present invention will become apparent from the following description and accompanying drawings. [Brief explanation of the drawings]

[0008] [Figure 1] Figure 1 is a schematic diagram illustrating the test protocol for evaluating the stability of various formulations of PDSP solutions. Combinations of PVP and PVA (hereinafter referred to as "PVP / PVA") were mixed to create different PDSP solutions at selected mixing ratios according to the study design. Part I: Selection of different PVP / PVA ratios. The pH of the PDSP solutions was adjusted with 1N HCl or 2N NaOH, and the solutions were filtered through 0.2 μm syringe filters. Each solution was then placed in a 20 ml screw-cap glass bottle. The filtered PDSP solutions were stirred at 1,100 RPM at room temperature. Aliquots of 80 μl of the PDSP solutions were taken at various time points to measure the size distribution profiles of small particles in the various formulation solutions using dynamic light scattering (DLS). The PDSP solutions were continuously stirred, and 300 μl aliquots of the PDSP solutions were further examined for precipitates 48 h after the start of stirring by centrifugation at 13,000 rpm. Part II: Particulate matter analysis. PDSP solutions with various PVP / PVA ratio formulations were filtered through a 0.2 μm syringe filter and each solution was placed in a 50 ml glass bottle with a screw cap. The filtered PDSP solutions were stirred at 390 RPM at room temperature for 24 hours. The United States Pharmacopeia (USP) <789> The formulation solution was tested for particulate matter according to the USP test. If the average number of particles present in the tested units does not exceed the appropriate value listed in Table 1, the solution complies with the USP <789> Pass the exam. [Table 1]

[0009] [Figure 2]Figure 2 shows the results of a stability test of PDSP formulations prepared with different PVP / PVA ratios (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 precipitates in the formulations with different PVP / PVA ratios containing 0.03% PDSP appeared as peaks to the right of the main peak in these charts.

[0010] [Figure 3] Figure 3 shows the stability test of PDSP formulations prepared with different PVP / PVA ratios under continuous running conditions. These solutions, with or without 0.03% PDSP, were continuously stirred at 1,100 RPM at room temperature. These solutions were examined by DLS after 0, 3, 5, 8, 24, and 48 hours, and the particle size profile was recorded. Finally, the solutions were centrifuged and examined after 48 hours to assess particle formation. Detailed Description

[0011] Embodiments of the present invention relate to formulations of PEDF-derived short peptides (PDSPs) with enhanced stability. Various 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, alopecia and / or hair depigmentation, osteoarthritis, skin aging, liver cirrhosis, or eye diseases or conditions. Examples of such PDSPs include those listed in Table 2. [Table 2]

[0012] According to embodiments of the invention, the PDSP may be SEQ ID NO: 1, 2, 3, 5, 6, 8, or 9. Additionally, the N-terminus of these peptides may optionally be protected by acylation (e.g., acetyl or propionyl protection), and the C-terminus may optionally be protected as an amide.

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

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

[0015] Polyvinylpyrrolidone (PVP) is a water-soluble, inert, non-toxic, pH-stable, biocompatible polymer that aids in the solubilization of both hydrophilic and lipophilic drugs. These advantages allow PVP to be used as a versatile excipient in pharmaceutical formulation development. Polyvinyl alcohol (PVA) is a water-soluble polymer that is often used to increase the viscosity of pharmaceuticals and as a lubricant and protectant in ophthalmic preparations. The use of a PVP / PVA combination has been found to have a soothing effect in eye drops. This combination has been investigated for its ability to provide long-term stability for PDSP formulations. Embodiments of the present invention demonstrate that the stability of PDSP solutions may be enhanced at specific PVP / PVA ratios.

[0016] The following are specific examples to illustrate embodiments of the present invention. However, those skilled in the art will recognize that these examples are for illustrative purposes only, and that 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 illustrative purposes, other PDSPs may be used instead. 1. Preparation of Different PVP / PVA Ratio Formulations

[0017] PVP / PVA formulations can be used at any suitable concentration, e.g., 0.5-10% w / v, preferably 1-5% w / v, and more preferably 2-4% w / v. PVP and PVA are readily available from commercial sources (e.g., Sigma-Aldrich). PVP suitable for use in embodiments of the present invention may have a number-average molecular weight ranging from 10-400 kDa, preferably 10-100 kDa, and more preferably 20-60 kDa. PVA suitable for use in embodiments of the present invention may have a weight-average molecular weight ranging from 10-100 kDa, preferably 10-50 kDa, and more preferably 10-30 kDa.

[0018] As an example, the following describes the formulation of a 2% w / v PVP / PVA formulation. Those skilled in the art will understand that these examples are for illustrative purposes only, and that other percentages (e.g., 3%, 4%, etc.) and / or PVP / PVA ratios can be similarly prepared. 2% PVP / PVA solutions with different ratios were prepared as follows: First, solutions of 2% PVP (number-average MW approximately 60,000) and 2% PVA (weight-average MW approximately 13,000) were prepared in distilled water. Then, for testing, they were physically mixed in ratios of 10 / 0, 8 / 2, 6 / 4, 4 / 6, 2 / 8, and 0 / 10. For example, for an 8 mL solution with a 6 / 4 PVP / PVA ratio, the following amounts of materials were prepared: 8mL, 2%PVP / PVA ratio=6 / 4:4.8mL, 2%PVP+3.2mL, 2%PVA

[0019] Various tonicity modifiers have been used in eye drops, including citrate (molecular weight 192.12), sodium chloride (molecular weight 58.44), sorbitol (molecular weight 182.17), and histidine (molecular weight 155.15). To evaluate their ability to ensure long-term stability, tonicity modifiers are sometimes added to PVP / PVA formulations to prepare a range of ophthalmic formulations. Examples are shown below. 1. Various 2% PVP / PVA ratio formulations containing citrate buffer (55 mM citric acid with 45 mM NaCl, pH 7.5). 2. Various 2% PVP / PVA ratio formulations containing citrate buffer (55 mM citric acid with 137 mM sorbitol, pH 7.5). 3. Various 2% PVP / PVA ratio formulations containing histidine buffer (125 mM histidine with 137 mM sorbitol, pH 7.6). 2. Preparation of PDSP in Various Formulations

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

[0021] The pH value of the formulation was measured after the PDSP was completely dissolved in the solution and then adjusted to 7.5 or 7.6 according to the test plan. Before use in the test, the PDSP formulation solution was filtered with a syringe filter (e.g., 0.2 μm filter). 3. Evaluation of shear resistance for PDSPs prepared with different formulations

[0022] We found that previous formulations of PDSP in citrate buffer were not stable during long-term storage. To test the effect of different PVP / PVA blends on stability, various PDSP formulations were subjected to stress conditions (e.g., shear stress) to accelerate changes.

[0023] Study Part I: 29-mer PDSP solutions (shown in Table 3) prepared with different PVP / PVA ratios, buffers, and excipients were stirred at 1,100 RPM at room temperature. At different time points, 80 μl of the PDSP solution was collected in a 1.5 ml Eppendorf tube, and dynamic light scattering (DLS) was used to measure the size distribution profiles of small particles in the different formulations. The PDSP solutions were stirred continuously, and after 48 hours of stirring, 300 μl of the PDSP solution was centrifuged at 13,000 rpm to further investigate the precipitates.

[0024] Part II: For particulate matter analysis, various PVP / PVA ratio blends of PDSP solutions were stirred at 390 RPM for 24 hours at room temperature. The blended solutions were then analyzed using USP <789> The formulation was evaluated according to the USP protocol. If the average number of particles present in the unit did not exceed the appropriate value, the formulation met the USP <789> The test was passed. The experimental procedure is shown in Figure 1. [Table 3] result Shear resistance of 29mer PDSP prepared in different PVP / PVA ratio solutions (10 / 0, 8 / 2, 6 / 4, 4 / 6, 2 / 8, 0 / 10, respectively) containing 1.55 mM citric acid and 137 mM sorbitol (pH 7.5).

[0025] The stability of PDSP prepared with formulations of different PVP / PVA ratios under stirring conditions was investigated using DLS and centrifugation analysis as screening tools to determine the resistance of these formulations to shear forces. The DLS results, shown in Figure 2, indicate that for the PDSP formulation with a PVP / PVA ratio of 8 / 2, the primary particle size distribution was consistently between 5 nm and 100 nm at various time points. This suggests that the formulation with a PVP / PVA ratio of 8 / 2 was the best among the test samples in providing protection against the effects of shear forces. In contrast, all other PVP / PVA ratios were less effective at protecting the PDSP formulation from shear forces, as evidenced by the formation of significant precipitates / aggregates.

[0026] In aqueous solutions, precipitation occurs through a process in which dissolved PDSP transforms from a formulation solution into an insoluble solid under stress conditions. After 48 hours of stirring, precipitation analysis based on high-speed centrifugation pellet formation showed that no precipitation was observed in the formulation with a PVP / PVA ratio of 8 / 2. In contrast, significant precipitation was observed in the other formulations (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 discussed in later sections, different PVP / PVA ratios may be preferred when using different concentrations or different buffers and / or tonicity agents. 2. Particulate matter analysis of PDSP Formulations containing different excipients with a PVP / PVA ratio of 8 / 2 under stirring conditions

[0027] Eye drops must be free of particulate matter visible on visual inspection. USP <789> describes a test for counting particles in a specific size range. For various PDSP formulations using different excipients and a PVP / PVA ratio of 8 / 2, USP <789> Specific substances were evaluated according to the test. The initial formulation was a PDSP formulation with a PVP / PVA ratio of 8 / 2, 55 mM citric acid, 137 mM sorbitol, and a pH of 7.5. However, this formulation was not suitable for PDSP formulation development because it required a large amount of pH adjustment to pH 7.5. Therefore, other PDSP formulations with a PVP / PVA ratio of 8 / 2 and other tonicity modifiers were investigated.

[0028] USP <789> Following the particulate matter test in ophthalmic solutions, various PDSP formulations containing other tonicity agents were evaluated under stirring conditions at a PVP / PVA ratio of 8 / 2. The results are shown in Table 4. Because the results for the formulation using 45 mM NaCl were not reproducible, the formulation with a PVP / PVA ratio of 8 / 2, 125 mM histidine, 137 mM sorbitol, and pH 7.6 was determined to be optimal for PDSP eye drops. [Table 4] 1. The three sets of tests were performed twice to confirm the results, and the two formulations gave identical results. 2. Substandard 3. Shear stress resistance of short peptides derived from different PEDFs prepared in solutions with different PVP / PVA ratios of 2% and 4%, respectively.

[0029] The experiments for the PVP / PVA-based formulation study described above were conducted using a 29-mer synthetic peptide, SEQ ID NO: 3. To investigate whether other PEDF-derived short-chain peptides could also be protected from agitation forces by PVP / PVA-based formulations, other PDSP peptides, such as a 39-mer (SEQ ID NO: 1) and a 20-mer (SEQ ID NO: 6), were prepared with different PVP / PVA ratios, as shown in Table 3. In Part II of the study, these other PDSPs were tested under the same shear stress load. The results for the 39-mer and 20-mer are shown in Table 5. Taken together, these results suggest that in addition to the PVP / PVA ratio of 8 / 2, formulations with other PVP / PVA ratios (PVP / PVA-based solutions containing 125 mM histidine and 137 mM sorbitol, pH 7.6) can provide protection against shear stress even for longer peptides (e.g., the 39-mer), and that PVP / PVA formulations containing different PDSPs (e.g., SEQ ID NOs: 1, 2, 3, 5, 6, 8, and 9) also exhibit improved stability.

[0030] Compared with 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] *Non-standard

[0031] The above examples clearly demonstrate that the new formulation of PDSP in various PVP / PVA-based solutions containing histidine or other tonicity agents dramatically improves stability compared to the original formulation in a citrate-based buffer.

[0032] Further experiments were conducted to investigate the effect of different PVP / PVA concentrations on the stability of PDSP formation. As shown in Table 6 below, for 4% PVP / PVA, better results were obtained with 4:6 and 2:8 PVP / PVA ratios. These results demonstrate that different concentrations and different PVP / PVA ratios can be selected to suit different applications in accordance with embodiments of the present invention. [Table 6] # For comparison, 0.1 mM PDSP was added to 350 mM nicotinamide, 20 mM histidine (without PVP / PVA). * Non-standard

[0033] Further experiments were performed to investigate 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 PDSP formulations. As shown in Table 7 below, PDSP at concentrations between 0.1 and 0.4 mM in PVP / PVA (4:6, 125 mM histidine, 130 mM sorbitol) all showed sufficient stability (USP 1000) after 24 hours of forced aggregation (390 rpm agitation) at room temperature. <789> These results demonstrate that the PVP / PVA formulation of the present invention can improve the stability of PDSP solutions at various concentrations for therapeutic use. [Table 7] # For comparison, PDSP 0.1 mM in 350 mM nicotinamide, 20 mM histidine (no PVP / PVA). PVP K30: Sigma [Cat. No. 81420] Average molecular weight = 40,000. PVA Sigma [Cat. No. 348406] Average molecular weight = 13,000-23,000, 98% hydrolyzed.

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

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

Claims

1. 1. An aqueous formulation comprising: a short peptide (PDSP) derived from pigment epithelium-derived factor (PEDF); a mixture of polyvinylpyrrolidone (PVP) and polyvinyl alcohol (PVA); an isotonicity agent; 1. An aqueous formulation comprising:

2. 2. The aqueous formulation of claim 1, wherein the PDSP is 14 to 39 amino acids in length.

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

4. 2. The aqueous formulation of claim 1, wherein the mixture of PVP and PVA has a PVP / PVA ratio of 8 / 2, 6 / 4, 4 / 6 or 2 / 8.

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

0.

6. 2. The aqueous formulation of claim 1, wherein the tonicity agent is a non-ionic tonicity agent.

7. 6. The aqueous formulation of claim 5, wherein the non-ionic tonicity agent is glycerin, sucrose, mannitol, or sorbitol.

8. 6. The aqueous formulation of claim 5, wherein the non-ionic tonicity agent is sorbitol.

9. 10. The aqueous formulation of claim 1, further comprising histidine.

10. 2. The aqueous formulation of claim 1, wherein the concentration of the PDSP is 0.01% to 1% w / v.

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