Compositions containing PEDF-derived short peptides (PDSPs) and their use

Abstract

The aqueous formulation contains a PEDF-derived short peptide (PDSP) having one of the sequences of SEQ ID NOs: 1, 2, 3, 5, 6, 8, or 9, histidine at a concentration of 1 mM to 100 mM, an antioxidant, and optionally a non-ionic isotonicity agent. The pH value is about 5 to 9. The antioxidant is nicotinamide at a concentration of 50 mM to 1000 mM. The non-ionic isotonicity agent is sorbitol at a concentration of 0 mM to 500 mM. The concentration of PDSP is 0.01% to 1% w / v.

Description

Technical Field

[0001] The present invention relates to compositions of short peptides derived from PEDF, and more particularly to formulations of such peptides and their use.

Background Art

[0002] Human pigment epithelium-derived factor (PEDF) is a secreted protein consisting of 418 amino acids with 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 found to have different functions. For example, the 34mer fragment (residues 44 - 77 of PEDF) has been confirmed to have anti-angiogenic activity, while the 44mer fragment (residues 78 - 121 of PEDF) has been confirmed to have neurotrophic properties.

[0003] Human PEDF-derived short peptides (PDSPs) have been found to be promising therapeutic agents for treating or preventing various diseases or disorders. For example, PDSPs are effective in promoting muscle regeneration or angiogenesis (U.S. Patent No. 9,884,012), treating alopecia and / or hair loss (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 conditions (e.g., retinal degeneration, Meibomian gland diseases, dry eye). The corresponding mouse PEDF-derived short peptides (moPDSPs) have also been found to have the same therapeutic effects. However, preparations of these peptides have been found to lack long-term stability. Therefore, a good formulation as this promising biopharmaceutical is needed.

Summary of the Invention

[0004] Embodiments of the present invention relate to formulations of short peptides (PDSPs) derived from PEDF, including SEQ ID NO: 1 (39mer), SEQ ID NO: 2 (34mer), SEQ ID NO: 3 (29mer), SEQ ID NO: 5 (24mer), SEQ ID NO: 6 (20mer), SEQ ID NO: 8 (mo29mer), and SEQ ID NO: 9 (mo20mer), where mo29mer and mo20mer are mouse PDSPs corresponding to human 29mer and 20mer, respectively.

[0005] One aspect of the present invention relates to an aqueous formulation comprising a PDSP having one of the sequences of SEQ ID NOs: 1, 2, 3, 5, 6, 8, or 9, histidine in a concentration of 1 mM to 100 mM, an antioxidant, and optionally a nonionic isotonic agent. The antioxidant is ascorbic acid or nicotinamide. The nonionic isotonic agent is sorbitol, dextrose, glycerin, mannitol, potassium chloride, sodium chloride, ethylene glycol, or propylene glycol.

[0006] According to some embodiments of the present invention, the pH value of the aqueous formulation may be about 5 to 9, preferably about 6.5 to 7.5. The nonionic isotonic agent is sorbitol, which is present in a concentration of 0 mM to 500 mM. The antioxidant is nicotinamide, which is present in a concentration of 50 mM to 1000 mM. The concentration of PDSP may be 0.01% to 1% w / v.

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

[0008] [Figure 1]This is a schematic diagram showing a test protocol for evaluating the stability of various formulations of PDSP solution. Various PDSP solutions were prepared according to the experimental design. The pH of the PDSP solution was adjusted with 1N HCl or 2N NaOH, filtered through a 0.2 μm syringe filter, and placed in a 50 ml glass bottle. The filtered PDSP solution was stirred at 1150 RPM at room temperature. Aliquots of 400 μl of the PDSP solution were taken at different time points (every 30 minutes until 7 or 9 hours had passed) and centrifuged at 13000 rpm to observe whether a precipitate appeared. The stirring of the PDSP solution was continued, and the precipitate was examined at 10, 12, 18, and 24 hours. The time of appearance of suspensions, precipitates, and turbidity was recorded.

[0009] [Figure 2] Figure 2 shows the results of stability tests on PDSP formulations prepared under continuous stirring conditions in 10 mM citrate buffer (pH 6.0) containing 0.85% NaCl and 20 mM histidine buffer (pH 7.0) containing different concentrations of nicotinamide. Each of these different formulations of PDSP was filtered and placed in a 50 mL beaker, then the solutions were stirred at 1150 RPM at room temperature. These solutions were examined every 30 minutes for the first 7 hours, and observation continued 12 hours after the start of stirring.

[0010] [Figure 3] Figure 3 shows the results of stability tests on PDSP formulations prepared using different concentrations of sorbitol in a 20 mM histidine solution / 150 mM nicotinamide solution. Stability tests were performed under continuous stirring. PDSPs prepared with eight different formulations were filtered and placed in 50 mL beakers, then the solutions were stirred at 1150 RPM at room temperature. These solutions were monitored every 30 minutes for the first 9 hours, and then at 12, 18, and 24 hours after the start of stirring. The time of precipitation and turbidity appearance was recorded.

[0011] [Figure 4]Figure 4 shows the time at which suspension, precipitation, and turbidity appeared in PDSP formulations prepared using different concentrations of sorbitol in 20 mM histidine solution / 150 mM nicotinamide solution under continuous stirring conditions. Curve 1: Time at which suspension appeared. Curve 2: Time at which visible precipitate appeared. Curve 3: Time at which turbid solution appeared. [Modes for carrying out the invention]

[0012] Embodiments of the present invention relate to formulations of short peptides (PDSPs) derived from PEDF with improved 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 arteriovenous formation, alopecia and / or hair loss, osteoarthritis, skin aging, cirrhosis, or eye diseases or conditions. Examples of such PDSPs are shown in Table 1. [Table 1]

[0013] According to embodiments of the present invention, the PDSP may be SEQ ID NOs: 1, 2, 3, 5, 6, 8, or 9. Furthermore, 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.

[0014] These PDSPs are prepared in citrate buffer and have been shown to be effective for therapeutic purposes in various preclinical studies. However, preparations of these short peptides (e.g., PDSP in 10 mM citrate buffer (pH 6.0) containing 0.85% w / v NaCl (SEQ ID NO: 3)) have been found to lack long-term stability (over several months).

[0015] Numerous factors, including chemical stress (e.g., oxidation, hydrolysis) and physical stress (e.g., temperature, light, and agitation), can affect the quality and stability of biopharmaceuticals, particularly during long-term storage. Accelerated stability testing was conducted to investigate the stability of different formulations of PDSP. Specifically, various formulations were tested under stress conditions, particularly shear stress, to identify the optimal formulation. After extensive testing, it was unexpectedly found that certain formulations exhibited superior long-term stability compared to the original citrate-buffered formulation.

[0016] The following describes specific examples to illustrate embodiments of the present invention. However, those skilled in the art will understand that these specific 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, the following examples use PDSP (SEQ ID NO: 3) for illustrative purposes, but other PDSPs may be used instead. 1. Citrate buffer (10 mM working citrate buffer containing 0.85% w / v NaCl, pH 6.0)

[0017] Citrate buffer solutions were prepared from citric acid and trisodium citrate to achieve the desired buffering capacity and pH. For example, solutions A and B were prepared using citric acid monohydrate (MW 210.14 kDa) (Merck) and trisodium citrate dihydrate (MW 294.12 kDa) (BioShop). Then, these two solutions were used to prepare citrate buffer solutions with the desired concentration and pH. The formulas for solutions A and B are as follows.

[0018] Solution A (0.1M citric acid monohydrate) (10 ml): 210.14 kDa × 10 / 1000 × 0.1 = 0.21 g citric acid monohydrate. Weigh 0.21 g of citric acid monohydrate and dissolve it in 10 ml of ddH2O to prepare 10 ml of Solution A stock.

[0019] Solution B (0.1 M trisodium citrate dihydrate) (10 ml): 294.12 kDa × 10 / 1000 × 0.1 = 0.294 g of trisodium citrate dihydrate. Weigh 0.294 g of citric acid monohydrate and dissolve it in 10 ml of ddH2O to produce 10 ml of Solution B stock.

[0020] To prepare a 10-fold citric acid buffer stock at pH 6.0, 1.15 ml of Solution A and 8.85 ml of Solution B were mixed to obtain 10 mL of 0.1 M citric acid buffer. Then, 10 ml of the 0.1 M citric acid buffer stock was diluted with 90 ml of ddH2O to produce 100 ml (1-fold solution) of 10 mM working citric acid buffer.

[0021] To prepare 10 mM citric acid buffer containing 0.85% w / v NaCl, 0.85 g of NaCl was added to 100 ml of 10 mM working citric acid buffer. Before use, the pH needs to be measured and adjusted based on the experimental design. 2. Histidine buffer (20 mM histidine buffer containing 0 - 260 mM sorbitol and / or 150 - 350 mM nicotinamide, pH 7.0)

[0022] To prepare 20 mL of 20 mM histidine buffer pH 7.0 for testing, 0.062 g of histidine and different weights of sorbitol and / or nicotinamide were dissolved in 15 mL of ddH2O. Examples of various preparations with different concentrations of sorbitol and nicotinamide are prepared using the following compositions shown in Table 2.

Table 2

[0023] The pH value of the buffer was adjusted to pH 7.0 using 2N NaOH or 1N HCl. The volume of 2N NaOH or 1N HCl used for pH adjustment was recorded, and then ddH2O was added to make the total volume 20 ml. 3. Preparation of PDSP of different formulations

[0024] The PDSP used in these examples is a short synthetic peptide (29mer) with an acetylated NH2 terminus and an amide at the COOH terminus. The molecular weight of PDSP is 3243.6 kDa. PDSP was dissolved in each of the above solutions at specific concentrations.

[0025] For example, to prepare 20 ml of PDSP solution in histidine / nicotinamide buffer or citrate buffer, 6.772 mg of the peptide product was added to 20 ml of histidine / nicotinamide buffer or citrate buffer.

[0026] After the PDSP was completely dissolved in the solution, the pH of the PDSP solution was measured and then adjusted to 7.0 or 6.0 according to the experimental design. Before use, each PDSP solution was filtered through a 0.2 μm syringe filter. 4. Stability evaluation of PDSPs in different formulations

[0027] The inventors noticed that previous formulations of PDSP in citrate buffer were not stable during long-term storage (over several months). To test the effect of different formulations on stability, various PDSP formulations were subjected to stress conditions (e.g., shear stress) to accelerate changes.

[0028] For these tests, 20 ml of PDSP prepared in different buffers and excipients (shown in Table 3) were filtered and placed in 50 ml beakers. The solutions were then stirred at 1150 RPM at room temperature. Aliquots of 400 μl of PDSP solution were taken into 1.5 ml Eppendorf tubes every 30 minutes for a maximum of 7 or 9 hours. The collected samples were centrifuged at 13000 rpm for 5 minutes to assess whether precipitation occurred. After continuous observation, stirring of the PDSP solution was continued for 24 hours. The appearance of the solution and the presence of precipitation were examined after stirring at 10, 12, 18, and 24 hours. The time of appearance of suspensions, precipitates, and turbidity was recorded. The experimental procedure is shown in Figure 1. [Table 3] result 1. Shear resistance of PEDF-derived short peptides (PDSPs) prepared in 10 mM citrate buffer (pH 6.0) containing 0.85% w / v NaCl.

[0029] The original formulation for PDSP preparation was a 10 mM citrate buffer (pH 6.0) containing 0.85% w / v NaCl. This formulation was suitable for various preclinical studies. However, this formulation developed turbidity over long-term storage (several months). Therefore, its stability was investigated using a forced agglutination method to elucidate its ability to resist shear forces. As shown in Figure 2, the solution was clear and transparent before stirring (Figure 2, top panel). In this formulation, suspensions were observed approximately 1 hour after the start of stirring (Figure 2, left panel and Table 4). Precipitation was observed 1.5 hours after the start of stirring, and a turbid solution was observed 2.5 hours later. These observations will be used as a baseline for comparison with other formulations. [Table 4] TIFF0007847082000005.tif1421662. Shear resistance of PEDF-derived short peptides (PDSPs) prepared with histidine buffer containing nicotinamide.

[0030] To investigate the effect of the antioxidant nicotinamide on the stability of PDSP in histidine-based buffers, PDSP prepared in 20 mM histidine buffer (pH 7.0) containing 150 mM, 300 mM, or 350 mM nicotinamide was selected as a comparison. As shown in Figure 2 and Table 4, suspensions were observed in PDSP prepared in 20 mM histidine buffer containing 150 mM, 300 mM, and 350 mM nicotinamide, respectively, at 5 hours, 4.5 hours, and 14 hours after the start of stirring (Table 4). The suspensions in the formulations containing histidine / nicotinamide buffer appeared significantly later compared to the formulations in citrate buffer.

[0031] In the experiment, the suspension in PDSP prepared with 10 mM citrate buffer containing 0.85% NaCl was found to be coarse, and small particles or fibers could be observed under a dissecting microscope. However, the suspension in the PDSP preparation prepared with histidine / nicotinamide buffer was extremely fine, with no visible particles under a dissecting microscope, and only reduced the clarity of the solution.

[0032] To evaluate whether precipitation could occur due to the presence of suspensions, 400 μl aliquots of each PDSP solution were taken into 1.5 ml Eppendorf tubes for centrifugation (13000 rpm, 5 minutes). As shown in Figure 2 (center panel) and Table 4, visible precipitation was observed at 5.5 hours, 4.5 hours, and 14.5 hours after the start of stirring for PDSP prepared in 20 mM histidine buffer containing 150 mM, 300 mM, and 350 mM nicotinamide, respectively.

[0033] After suspension or precipitate was observed, stirring was continued until the PDSP solution became cloudy. Figure 2 (bottom panel) and Table 4 show that PDSP formulations prepared with 20 mM histidine buffer containing 150 mM, 300 mM, and 350 mM nicotinamide became cloudy at 13.5 hours, 7–24 hours, and 16.5 hours, respectively, after the start of stirring.

[0034] Compared to PDSP preparations prepared with citrate buffer, PDSP preparations prepared with histidine / nicotinamide buffer exhibited better resistance to shear stress. Furthermore, among these histidine / nicotinamide preparations, the solution containing 350 mM nicotinamide showed a longer precipitation time than the solutions containing 150 mM and 300 mM nicotinamide, suggesting that higher concentrations of nicotinamide can increase the stability of PDSP in preparations prepared with histidine-based buffers. 3. Resistance to shear force of PEDF-derived short peptides (PDSPs) prepared with histidine / nicotinamide buffer containing different concentrations of sorbitol.

[0035] It has been reported that administering nicotinamide to the eyes may cause eye irritation (Keri, G. 2005. Reassessment of the one experiment from the requirement of the tolerance for nicotinamide, U.S. Environmental Protection Agency, Washington DC 20460.1-12). Therefore, sorbitol was used to replace all or part of the nicotinamide in the histidine-based buffer. As shown in Table 4, in the formulation of 20 mM histidine / 260 mM sorbitol alone, suspension was observed after only 3 hours of stirring. This suspension was found at 3 hours, 4.5 hours, 4 hours, 13 hours, 13.5 hours, 6 hours, 5 hours, and 4.5 hours, respectively, after the start of stirring in PDSP prepared with 20 mM histidine / 150 mM nicotinamide buffer containing 120 mM, 125 mM, 130 mM, 140 mM, 150 mM, 160 mM, 170 mM, and 180 mM sorbitol (Figures 3, 4, and Table 4).

[0036] Among these histidine / nicotinamide preparations, suspensions, precipitates, and turbidity were observed after 13 hours of continuous stirring in PDSP prepared with 20 mM histidine / 150 mM nicotinamide containing 140 mM and 150 mM sorbitol, suggesting that the optimal concentration of sorbitol in histidine / nicotinamide preparations may be in the range of approximately 140 mM to 150 mM (Figures 3, 4, and Table 4). Furthermore, the stability of PDSP prepared with 20 mM histidine / 150 mM nicotinamide / 140 or 150 mM sorbitol buffer and PDSP prepared with 20 mM histidine / 350 mM nicotinamide buffer alone was equivalent, suggesting that sorbitol can be used to replace a portion of the nicotinamide.

[0037] These results, along with the data above, suggest that formulations containing histidine / nicotinamide exhibit superior stability of PDSP compared to citrate / NaCl formulations. Precipitation appeared 1 hour after the start of stirring for PDSP prepared with 10 mM citrate containing 0.85% NaCl, but was observed after 5 hours of stirring for PDSP prepared with 20 mM histidine containing 150 mM to 350 mM nicotinamide. The five-fold longer duration of precipitation in histidine / nicotinamide formulations indicates that PDSP is dramatically more stable in histidine / nicotinamide formulations compared to citrate / NaCl formulations. Furthermore, between formulations with different nicotinamide concentrations, no precipitation was observed until 14.5 hours after continuous stirring, indicating that higher concentrations of nicotinamide are more suitable for maintaining the stability of the excipient, PDSP.

[0038] Compared to PDSP preparations prepared in citrate buffer, PDSP preparations prepared in histidine / sorbitol-only buffer exhibit a better ability to maintain PDSP stability. However, compared to preparations prepared in histidine / nicotinamide-only buffer, the ability of histidine / sorbitol-only preparations to maintain PDSP stability is still not sufficiently good, indicating that nicotinamide may be an important component for maintaining PDSP stability in histidine-based buffers.

[0039] When a portion of the nicotinamide was substituted with an isotonic agent such as sorbitol, the time to precipitate formation was similar for PDSP prepared with 20 mM histidine / 350 mM nicotinamide (14.5 hours) and PDSP prepared with 20 mM histidine / 150 mM nicotinamide / 140 mM or 150 mM sorbitol (13 and 14 hours, respectively). These data further support the idea that a concentration of approximately 140–150 mM is a better choice for sorbitol concentration in PDSP formulations prepared with 20 mM histidine / 150 mM nicotinamide buffer.

[0040] Overall, the formulations tested by the inventors demonstrated that histidine / nicotinamide is a far better base buffer for formulations containing PDSP (e.g., PDSP; SEQ ID NO: 3) than citrate buffer. According to embodiments of the present invention, PDSP may be at any suitable concentration (e.g., 0.01% to 5% w / v, preferably 0.01% to 1% w / v), and the histidine buffer may be used at any suitable concentration, e.g., 1 mM to 100 mM, preferably 5 mM to 60 mM, more preferably 10 mM to 40 mM, and most preferably 15 mM to 30 mM. The pH of the formulation may be in the range of 5 to 9, preferably about neutral, e.g., 6.5 to 7.5, and most preferably about 7.0. The formulation contains an antioxidant, preferably nicotinamide, at an appropriate concentration, for example, 50 mM to 1000 mM, preferably 100 mM to 700 mM, more preferably 200 mM to 500 mM, and most preferably 300 mM to 400 mM. For example, a preferred formulation of PDSP solution may contain 20 mM histidine (pH 7.0) containing 350 mM nicotinamide. The formulation may also contain a nonionic isotonic agent, preferably sorbitol, at an appropriate concentration, for example, 0 mM to 500 mM, preferably 10 mM to 400 mM, more preferably 50 mM to 300 mM, and most preferably 100 mM to 200 mM. For example, a preferred formulation of PDSP solution may contain 20 mM histidine (pH 7.0) containing 150 mM nicotinamide and 150 mM sorbitol.

[0041] The formulations of the present invention can be used to treat various diseases and conditions, such as retinal degeneration, meibomian gland diseases, and dry eye. For administration to the eye, the formulations may be eye drops.

[0042] Embodiments of the present invention are illustrated using a limited number of examples. Those skilled in the art will understand that these embodiments are for illustrative purposes only and do not imply any limitation of the scope of the invention, as other modifications and variations are possible without departing from the scope of the invention. Accordingly, the scope of the invention should be limited by the appended claims.

Claims

1. A short peptide (PDSP) derived from PEDF having the sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 9, Antioxidants, Contains histidine at concentrations of 10 mM to 40 mM. A aqueous formulation, The antioxidant is nicotinamide at a concentration of 150 mM to 350 mM. Aqueous formulation.

2. The pH value is between 5 and 9. The aqueous formulation according to claim 1.

3. Further contains a nonionic isotonic agent The aqueous formulation according to claim 1.

4. The nonionic isotonic agent is sorbitol. The aqueous formulation according to claim 3.

5. The sorbitol concentration is between 120 mM and 500 mM. The aqueous formulation according to claim 4.

6. The PDSP has the sequence of sequence number 3 The aqueous formulation according to any one of claims 1 to 5.

7. The concentration of the aforementioned PDSP is 0.01% to 1% w / v. The aqueous formulation according to claim 6.

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