Compositions comprising PEDF-derived short peptides (PDSP) and uses thereof

Abstract

Formulations of short peptides derived from PEDF are provided.SOLUTION: The aqueous formulation comprises a PEDF-derived short peptide (PDSP) having a specific sequence, histidine at a concentration of 1mM to 100mM, an antioxidant, and optionally a non-ionic isotonic agent. The pH value is about 5-9. The antioxidant is nicotinamide, which is at a concentration between 50mM and 1000mM. The non-ionic tonicity agent is sorbitol, which is at a concentration between 0mM and 500mM. The concentration of PDSP is from 0.01% to 1% w / v.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to compositions of short peptides derived from PEDF, and 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 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 shown to serve different functions. For example, a 34-mer fragment (residues 44-77 of PEDF) has been identified as having anti-angiogenic activity, while a 44-mer fragment (residues 78-121 of PEDF) has been identified as having neurotrophic properties.

[0003] Short peptides derived from human PEDF (PDSPs) have been shown to be promising therapeutic agents for treating or preventing various diseases or disorders. For example, PDSPs have been shown to be effective in promoting muscle regeneration or arteriogenesis (U.S. Pat. No. 9,884,012), treating alopecia and / or baldness (U.S. Pat. No. 9,938,328), treating osteoarthritis (U.S. Pat. No. 9,777,048), preventing or improving skin aging (U.S. Pat. No. 9,815,878), treating liver cirrhosis (U.S. Pat. No. 8,507,446), or treating various eye diseases or conditions (e.g., retinal degeneration, meibomian gland disease, dry eye). The corresponding short peptides derived from mouse PEDF (moPDSPs) have also been shown to have the same therapeutic effects. However, preparations of these peptides have been found to lack long-term stability. Therefore, improved formulations of this promising biopharmaceutical are needed. Summary of the Invention

[0004] An embodiment of the present invention relates to formulations of PEDF-derived short peptides (PDSPs), 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 murine PDSPs corresponding to the human 29mer and 20mer, respectively.

[0005] One aspect of the present invention relates to an aqueous formulation comprising a PDSP having one 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 tonicity agent. The antioxidant is ascorbic acid or nicotinamide. The non-ionic tonicity 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 aqueous formulation may have a pH value of about 5 to 9, preferably about 6.5 to 7.5. The non-ionic isotonic agent is sorbitol at a concentration of 0 mM to 500 mM. The antioxidant is nicotinamide at 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 accompanying drawings. [Brief explanation of the drawings]

[0008] [Figure 1]FIG. 1 is a schematic diagram showing the test protocol for evaluating the stability of various formulations of PDSP solution. Various PDSP solutions were prepared according to the experimental design. The pH value 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) and centrifuged at 13000 rpm to observe whether precipitate appeared. The PDSP solution was continuously stirred and examined for precipitate at 10, 12, 18, and 24 hours. The time for the appearance of suspension, precipitate, and turbidity was recorded.

[0009] [Figure 2] Figure 2 shows the results of a stability test of 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. The PDSP prepared in these different formulations was placed in a 50 mL beaker after filtration, and the solution was then stirred at 1150 RPM at room temperature. The solutions were examined every 30 minutes for the first 7 hours, and continued to be observed for 12 hours after the start of stirring.

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

[0011] [Figure 4]Figure 4 shows the time to appearance of suspension, precipitation, and turbidity under continuous stirring conditions for PDSP formulations prepared with different concentrations of sorbitol in a 20 mM histidine / 150 mM nicotinamide solution. Curve 1: Time when suspension appeared. Curve 2: Time when visible precipitation appeared. Curve 3: Time when a turbid solution appeared. DETAILED DESCRIPTION OF THE INVENTION

[0012] Embodiments of the present invention relate to formulations of short peptides derived from PEDF (PDSPs) 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 arteriogenesis, alopecia and / or hair loss, osteoarthritis, skin aging, liver cirrhosis, or eye diseases or conditions. Examples of such PDSPs include those listed in Table 1. [Table 1]

[0013] According to embodiments of the present 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.

[0014] These PDSPs are formulated 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 (SEQ ID NO: 3) in 10 mM citrate buffer (pH 6.0) containing 0.85% w / v NaCl) have been found to lack long-term stability (over several months).

[0015] 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 biopharmaceuticals, especially during long-term storage. To investigate the stability of different formulations of PDSP, accelerated stability studies were conducted. Specifically, various formulations were tested under stress conditions, particularly shear stress, to identify the optimal formulation. After extensive testing, certain formulations were unexpectedly found to have superior long-term stability to the original citrate-buffered formulation.

[0016] Specific examples are provided below to illustrate embodiments of the present invention. However, those skilled in the art will appreciate that these specific examples are for illustrative purposes only, and that other changes and modifications are possible without departing from the scope of the present invention. For example, although the following examples use PDSP (SEQ ID NO: 3) for illustrative purposes, 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 buffer 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), respectively. These two solutions were then used to create citrate buffer solutions with the desired concentration and pH. The formulas for solutions A and B are as follows:

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

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

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

[0021] To prepare 10 mM citrate buffer containing 0.85% w / v NaCl, add 0.85 g of NaCl to 100 ml of 10 mM working citrate buffer. Before use, the pH should be measured and adjusted based on the experimental design. 2. Histidine buffer (20 mM histidine buffer containing 0 to 260 mM sorbitol and / or 150 to 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 weight amounts of sorbitol and / or nicotinamide were dissolved in 15 mL of ddH2O. Examples of various preparations with different sorbitol and nicotinamide concentrations 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 to adjust the pH value was recorded, and then ddH2O was added to bring the total volume to 20 ml. 3. Preparation of PDSP in Different Formulations

[0024] The PDSP used in these examples is a short synthetic peptide (29-mer) 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 a specific concentration.

[0025] For example, to prepare 20 ml of PDSP solution in histidine / nicotinamide buffer or citrate buffer, 6.772 mg of 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 value of the PDSP solution was measured and then adjusted to 7.0 or 6.0 according to the experimental design. Before use, the PDSP solutions were filtered through a 0.2 μm syringe filter, respectively. 4. Stability evaluation of PDSP in different formulations

[0027] The inventors have found that previous formulations of PDSP in citrate buffer are not stable upon 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 their transformation.

[0028] For these tests, 20 milliliters of PDSP prepared in different buffers and excipients (shown in Table 3) were placed in a 50-mL beaker after filtration. The solutions were then stirred at 1,150 RPM at room temperature. Aliquots of 400 μl of the PDSP solution were collected in 1.5-mL Eppendorf tubes every 30 minutes for up to 7 or 9 hours. The collected samples were centrifuged at 13,000 RPM for 5 minutes to assess whether precipitation had occurred. After continuous observation, stirring of the PDSP solution was continued for up to 24 hours. The appearance of the solution and the presence of precipitation were examined after 10, 12, 18, and 24 hours of stirring. The time for the appearance of suspension, precipitation, 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 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 (many months). Therefore, its stability was investigated using a forced flocculation 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, a suspension was observed approximately 1 hour after stirring began (Figure 2, left panel and Table 4). Precipitation was observed 1.5 hours after stirring began, and a turbid solution was observed 2.5 hours after stirring began. These observations serve as a baseline for comparison with other formulations. [Table 4] TIFF2026010112000005.tif1421662. Ability of PEDF-derived short peptides (PDSPs) prepared in histidine buffer containing nicotinamide to resist shear forces

[0030] To examine the effect of the antioxidant nicotinamide on the stability of PDSP in histidine-based buffers, PDSPs formulated in 20 mM histidine buffer (pH 7.0) containing 150 mM, 300 mM, or 350 mM nicotinamide were selected for comparison. As shown in Figure 2 and Table 4, suspensions were observed 5, 4.5, and 14 hours after the start of stirring for PDSPs formulated in 20 mM histidine buffer containing 150 mM, 300 mM, and 350 mM nicotinamide, respectively (Table 4). The onset of suspension in the histidine / nicotinamide buffer formulations was significantly delayed compared to the citrate buffer formulation.

[0031] In experiments, the suspension in PDSP prepared in 10 mM citrate buffer containing 0.85% NaCl was found to be coarse, with small particles or fibers visible under a dissecting microscope. However, the suspension in PDSP preparations prepared in histidine / nicotinamide buffer was very fine, with no visible particles under a dissecting microscope, which only reduced the transparency of the solution.

[0032] To assess whether the presence of suspended solids could also cause precipitation, 400 μl aliquots of each PDSP solution were collected in 1.5 ml Eppendorf tubes for centrifugation (13,000 rpm, 5 min). As shown in Figure 2 (center panel) and Table 4, visible precipitation was observed 5.5, 4.5, and 14.5 hours after stirring began for PDSP prepared in 20 mM histidine buffer containing 150 mM, 300 mM, and 350 mM nicotinamide, respectively.

[0033] After suspension or precipitation was observed, stirring was continued until the PDSP solution became cloudy. Figure 2 (lower panel) and Table 4 show that PDSP formulations prepared in 20 mM histidine buffer containing 150 mM, 300 mM, and 350 mM nicotinamide became cloudy 13.5 hours, 7 to 24 hours, and 16.5 hours after stirring began, respectively.

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

[0035] It has been reported that nicotinamide can cause ocular irritation when administered to the eye (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 histidine-based buffers. As shown in Table 4, in the 20 mM histidine / 260 mM sorbitol-only formulation, suspension was observed after only 3 hours of stirring. This suspension was observed in PDSP prepared in 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 at 3, 4.5, 4, 13, 13.5, 6, 5, and 4.5 hours after the start of stirring, respectively (Figure 3, Figure 4, and Table 4).

[0036] Among these histidine / nicotinamide formulations, suspension, precipitation, 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 range of approximately 140 mM to 150 mM may be the optimal concentration of sorbitol in histidine / nicotinamide formulations (Figure 3, Figure 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-only buffer was comparable, suggesting that sorbitol can be used to partially replace nicotinamide.

[0037] These results, along with the data above, suggest that the histidine / nicotinamide-containing formulation provides superior PDSP stability compared with the citrate / NaCl formulation. Precipitation appeared after 1 h of stirring for PDSP prepared in 10 mM citrate with 0.85% NaCl, whereas precipitation was observed after 5 h of stirring for PDSP prepared in 20 mM histidine with 150 mM–350 mM nicotinamide. The fivefold longer duration of precipitation for the histidine / nicotinamide formulation indicates dramatically greater stability of PDSP in the histidine / nicotinamide formulation compared with the citrate / NaCl formulation. Furthermore, precipitation was not observed for formulations containing different concentrations of nicotinamide until 14.5 h after continuous stirring, indicating that higher concentrations of nicotinamide are better suited to maintaining the stability of the excipient PDSP.

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

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

[0040] Overall, the formulations tested by the inventors demonstrated that histidine / nicotinamide is a much 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 used at any suitable concentration (e.g., 0.01% to 5% w / v, preferably 0.01% to 1% w / v), and 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 value of the formulation may range from 5 to 9, preferably about neutral, e.g., 6.5 to 7.5, most preferably about 7.0. The formulation contains an antioxidant, preferably nicotinamide, at a suitable concentration, e.g., 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 non-ionic isotonic agent, preferably sorbitol, at a suitable concentration, e.g., 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 a variety of diseases and conditions, such as retinal degeneration, meibomian gland disease, dry eye, etc. For administration to the eye, the formulations can be eye drops.

[0042] Embodiments of the present invention have been illustrated using a limited number of examples. Those skilled in the art will understand that these examples are for illustrative purposes only and are not meant to limit the scope of the present invention, as other changes and modifications are possible without departing from the scope of the present invention. Therefore, the scope of the present invention should be limited by the appended claims.

Claims

1. a PEDF-derived short peptide (PDSP) 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, and and histidine at a concentration of 1 mM to 100 mM.

1. An aqueous formulation comprising: the antioxidant is nicotinamide at a concentration of 50 mM to 1000 mM; Aqueous formulation.

Images