TWI823037B - Compositions comprising pedf-derived short peptides (pdsp) and uses thereof - Google Patents

Abstract

An aqueous formulation includes a PEDF-derived short peptide (PDSP) having the sequence of one of SEQ ID NO: 1, 2, 3, 5, 6, 8, or 9; histidine having a concentration of 1 mM – 100 mM; and an antioxidant and optionally a non-ionic tonicity agent. The pH value is around 5–9. The antioxidant is nicotinamide, which is at a concentration of 50 mM – 1000 mM. The non-ionic tonicity agent is sorbitol, which is at a concentration of 0 mM – 500 mM. A concentration of the PDSP is 0.01% - 1% w/v.

Description

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

The present invention relates to compositions of PEDF-derived short peptides, and in particular to formulations of such peptides and their uses.

Human pigment epithelium-derived factor (PEDF) is a secreted 418 amino acid protein with a molecular weight of approximately 50 kDa. PEDF is a multifunctional protein with many biological functions (see US Patent Application Publication No. 2010/0047212). It was found that different peptide regions of human PEDF are responsible for different functions. For example, a 34-mer fragment (residues 44 to 77 of PEDF) has been identified to have anti-angiogenic activity, while a 44-mer fragment (residues 78 to 121 of PEDF) has been identified to have neurotrophic properties.

Human PEDF-derived short peptides (PDSP) have been found to be promising therapeutic agents for the treatment or prevention of various diseases or conditions. For example, PDSP has been found to be effective in promoting muscle regeneration or arteriogenesis (U.S. Patent No. 9,884,012), treating alopecia and/or hair discoloration (U.S. Patent No. 9,938,328), and treating osteoarthritis (U.S. Patent No. 9,777,048 ), prevent or improve skin aging (U.S. Patent No. 9,815,878), treat liver cirrhosis (U.S. Patent No. 8,507,446), or treat various eye diseases or conditions (such as retinal degeneration, meibomian gland disease, dry eye syndrome). The corresponding mouse PEDF-derived short peptide (moPDSP) was also found to have the same therapeutic effect. However, formulations of these peptides were found to lack long-term stability. Therefore, there is a need for better formulations for such promising biopharmaceutical products.

Embodiments of the present invention relate to formulations of PEDF-derived short peptides (PDSP), including SEQ ID NO: 1 (39-mer), SEQ ID NO: 2 (34-mer), SEQ ID NO: 3 (29-mer) 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), wherein mo29 The mo20-mer and mo20-mer are mouse PDSP corresponding to the human 29-mer and 20-mer respectively.

One aspect of the invention relates to an aqueous formulation comprising a PDSP having the sequence of one of SEQ ID NO: 1, 2, 3, 5, 6, 8 or 9; having a range of 1 mM to 100 Histine at a concentration of 1.0 mM; and antioxidants and non-ionic tonicity agents if necessary. Antioxidants are ascorbic acid or nicotinamide. Nonionic tonicity agents are sorbitol, dextrose, glycerin, mannitol, potassium chloride, sodium chloride, ethylene glycol or propylene glycol.

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 tonicity agent is sorbitol, which is available in concentrations from 0 mM to 500 mM. The antioxidant is nicotinamide, which is available in concentrations from 50 mM to 1000 mM. The concentration of PDSP can be 0.01% to 1% w/v.

Other aspects of the present invention will be apparent from the following description and drawings.

Embodiments of the present invention are directed to formulations of PEDF-derived short peptides (PDSP) with enhanced stability. Various human PDSPs are found to be useful in the treatment or prevention of various diseases or conditions, including muscle regeneration or arterial angiogenesis, alopecia and/or hair discoloration, osteoarthritis, skin aging, liver cirrhosis, or eye diseases or conditions. Promising therapeutic agents. Examples of such PDSPs may include those shown in Table 1 : Table 1 : Examples of PEDF-derived short peptides (PDSPs) Name sequence SEQ ID NO Human PEDF residues 39-mer LSVATALSAL SLGAEQRTESIIHRALYYDL ISSPDIHGT 1 82 to 121 34-mer ALSAL SLGAEQRTESIIHRALYYDL ISSPDIHGT 2 88 to 121 29-mer SLGAEQRTESIIHRALYYDL ISSPDIHGT 3 93 to 121 25-mer EQRTESIIHRALYYDLISSPDIHGT 4 97 to 121 24mer SLGAEQRTESIIHRALYYDL ISSP 5 93 to 116 20-mer SLGAEQRTESIIHRALYYDL 6 93 to 112 18-mer EQRTESIIHRALYYDLIS 7 97 to 114 mo29mer SLGAEHRTESVIHRALYYDL ITNPDIHST 8 mouse mo20mer SLGAEHRTESVIHRALYYDL 9 mouse

According to embodiments of the present invention, the PDSP may be SEQ ID NO: 1, 2, 3, 5, 6, 8 or 9. In addition, the N-terminus of these peptides may be optionally protected by amide protection (eg, acetyl or propyl group protection), and the C-terminus may be optionally protected by amide protection.

These PDSPs have been prepared in citrate buffer and found to be effective for therapeutic purposes in various preclinical studies. However, formulations of these short peptides, such as PDSP (SEQ ID NO: 3) in 10 mM citrate buffer (pH 6.0) containing 0.85% w/v NaCl, were found to lack long-term stability (within several months ).

Many factors, including chemical stress (such as oxidation, hydrolysis, etc.) and physical stress (such as temperature, light and stirring), 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 performed. Specifically, various formulations are tested under stress conditions, particularly under shear stress, to identify optimal formulations. After extensive testing, it was unexpectedly found that certain formulations had better long-term stability than the original citrate buffer formulation.

Specific examples are described below to illustrate embodiments of the invention. However, those skilled in the art will understand that these specific examples are for illustration only and other modifications and changes may be made without departing from the scope of the present invention. For example, although the following examples are illustrated using PDSP (SEQ ID NO: 3), other PDSPs may be used instead. 1. Citrate buffer (10 mM citrate working buffer containing 0.85% w/v NaCl, pH 6.0)

Prepare citrate buffer from citric acid and trisodium citrate to achieve desired buffer capacity and pH. For example, citric acid monohydrate (MW 210.14 kDa) (Merck) and trisodium citrate dihydrate (MW 294.12 kDa) (BioShop) were used to prepare solution A and solution B, respectively. These two solutions are then used to prepare a citrate buffer with the desired concentration and pH. The formulas of solutions A and B are as follows:

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 in 10 ml of _ddH2O to produce 10 ml of Solution A stock.

Solution B (0.1M trisodium citrate dihydrate) (10 ml): 294.12 kDa × 10/1000 × 0.1 = 0.294 g trisodium citrate dihydrate. Weigh 0.294 g of trisodium citrate dihydrate and dissolve in 10 ml of _ddH2O to yield 10 ml of Solution B stock.

To prepare 10X Citrate Buffer Stock pH 6.0, mix 1.15 ml of Solution A and 8.85 ml of Solution B to obtain 10 mL of 0.1M Citrate Buffer. Then, 10 ml of the 0.1 M citrate buffer stock solution was diluted with 90 ml ddH ₂ O to produce 10 mM citrate working buffer, 100 ml (1X solution).

To prepare 10 mM Citrate Buffer containing 0.85% w/v NaCl, add 0.85 g NaCl to 100 ml of 10 mM Citrate Working Buffer. Before use, pH should be measured and adjusted based on study design. 2. Histidine buffer (20 mM histidine buffer containing 0 to 260 mM sorbitol and/or 150 to 350 mM nicotinamide, pH 7.0)

To prepare 20 mL of 20 mM histidine buffer pH 7.0 for testing, 0.062 g of histidine and varying weights of sorbitol and/or nicotinamide were dissolved in 15 mL of ddH ₂ O. Examples of preparation of various formulations with different sorbitol and nicotinamide concentrations using the following compositions shown in Table 2 : Table 2 : Various histidine buffer compositions 20 mM histidine/150 mM nicotinamide: 0.37 g/20 ml 20mM Histidine/300mM Nicotinamide: 0.73g/20ml 20 mM histidine/350 mM nicotinamide: 0.86 g/20 ml 20 mM histidine/120 mM sorbitol: 0.44 g/20 ml 20 mM histidine/140 mM sorbitol: 0.51 g/20 ml 20 mM histidine/160 mM sorbitol: 0.58 g/20 ml 20 mM histidine/180 mM sorbitol: 0.66 g/20 ml

Adjust the pH of the buffer to pH 7.0 using 2N NaOH or 1N HCl. Record the volume of 2N NaOH or IN HCl used for pH adjustment, and then add _ddH2O so that the total volume is 20 ml. 3. Preparation of PDSP in different formulations

The PDSP used in these examples is a short synthetic peptide (29-mer) that is acetylated at the NH _terminus and has an amide at the COOH terminus. The molecular weight of PDSP is 3243.6 kDa. PDSP was dissolved in each solution described above at a specific concentration.

For example, to prepare 20 ml of PDSP solution in Histidine/Nicotinamide or Citrate Buffer, add 6.772 mg of peptide product to 20 ml of Histidine/Nicotinamide Buffer or Citrate Buffer.

After the PDSP is completely dissolved in the solution, the pH value of the PDSP solution is measured, and then the pH value is adjusted to 7.0 or 6.0 according to the study design. Before use, filter each PDSP solution through a 0.2 µm syringe filter. 4. Stability evaluation of PDSP in different formulations

We noticed that earlier formulations of PDSP in citrate buffer were unstable during long-term storage (within months). To test the effect of different formulations on stability, various PDSP formulations were subjected to stress conditions (eg, shear stress) to accelerate change.

For these tests, twenty (20) ml of PDSP prepared in different buffers and excipients (as shown in Table 3 ) were each placed into 50 mL beakers after filtration. The solutions were then subjected to stirring at room temperature at 1,150 RPM. Every half hour up to 7 or 9 hours, collect aliquots of 400 µl of PDSP solution in 1.5 ml Eppendorf tubes. The collected samples were centrifuged at 13,000 rpm for 5 minutes to assess whether any precipitation occurred. After continuous observation, continue stirring the PDSP solution until 24 hours. After stirring for 10 hours, 12 hours, 18 hours and 24 hours, the solution appearance and possible precipitation were studied. Record the time of appearance of suspended matter, sedimentation and turbidity. The experimental procedure is illustrated in Figure 1 . Table 3. List of formulations tested in this study. Excipients base buffer pH value 0.85%NaCl 10 mM citrate buffer 6.0 150 mM Nicotinamide 20 mM Histidine Buffer 7.0 300 mM Nicotinamide 20 mM Histidine Buffer 7.0 350 mM Nicotinamide 20 mM Histidine Buffer 7.0 260 mM sorbitol 20 mM Histidine Buffer 7.0 150 mM Nicotinamide, 120 mM Sorbitol 20 mM Histidine Buffer 7.0 150 mM Nicotinamide, 125 mM Sorbitol 20 mM Histidine Buffer 7.0 150 mM Nicotinamide, 130 mM Sorbitol 20 mM Histidine Buffer 7.0 150 mM Nicotinamide, 140 mM Sorbitol 20 mM Histidine Buffer 7.0 150 mM Nicotinamide, 150 mM Sorbitol 20 mM Histidine Buffer 7.0 150 mM Nicotinamide, 160 mM Sorbitol 20 mM Histidine Buffer 7.0 150 mM Nicotinamide, 170 mM Sorbitol 20 mM Histidine Buffer 7.0 150 mM Nicotinamide, 180 mM Sorbitol 20 mM Histidine Buffer 7.0 Results 1. Resistance to shear forces of PEDF-derivatized short peptides (PDSP) prepared in 10 mM citrate buffer (pH 6.0) containing 0.85% w/v NaCl

The original formulation of the PDSP formulation was 10 mM citrate buffer (pH 6.0) containing 0.85% w/v NaCl. This formulation is suitable for a variety of preclinical studies. However, this formulation developed turbidity during long-term storage (several months). Therefore, the forced aggregation method was used to study its stability 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). Approximately 1 hour after starting stirring, suspended material was seen in this formulation ( Figure 2 , left panel and Table 4 ). Precipitate and turbid solution were observed 1.5 and 2.5 hours after starting stirring. These observations will be used as a baseline for comparison with other formulations. Table 4. Stability of PDSP prepared in different formulations under stirring conditions solution Suspension ( hours ) Precipitation ( hours ) Turbidity ( hours ) Excipients /pH base buffer 0.85% NaCl, pH 6.0 10 mM citrate buffer 1 1.5 2 solution Suspension ( hours ) Precipitation ( hours ) Turbidity ( hours ) Excipients /pH base buffer 150 mM Nicotinamide, pH 7.0 20 mM Histidine Buffer 5 5.5 13.5 300 mM Nicotinamide, pH 7.0 20 mM Histidine Buffer 4.5 4.5 between 7 and 24 350 mM Nicotinamide, pH 7.0 20 mM Histidine Buffer 14 14.5 16.5 260 mM Sorbitol, pH 7.0 20 mM Histidine Buffer 3 3 12 150 mM Nicotinamide, 120 mM Sorbitol, pH 7.0 20 mM Histidine Buffer 3 4 between 9 and 12 150 mM Nicotinamide, 125 mM Sorbitol, pH 7.0 20 mM Histidine Buffer 4.5 6.5 between 9 and 24 150 mM Nicotinamide, 130 mM Sorbitol, pH 7.0 20 mM Histidine Buffer 4 5 between 9 and 12 150 mM Nicotinamide, 140 mM Sorbitol, pH 7.0 20 mM Histidine Buffer 13 13 18 150 mM Nicotinamide, 150 mM Sorbitol, pH 7.0 20 mM Histidine Buffer 13.5 14 18 150 mM Nicotinamide, 160 mM Sorbitol, pH 7.0 20 mM Histidine Buffer 6 6 between 9 and 12 150 mM Nicotinamide, 170 mM Sorbitol, pH 7.0 20 mM Histidine Buffer 5 5 between 9 and 12 150 mM Nicotinamide, 180 mM Sorbitol, pH 7.0 20 mM Histidine Buffer 4.5 4.5 between 9 and 12 2. The shear resistance of PEDF-derived short peptides (PDSP) prepared in histidine buffer containing nicotinic acid.

To study the effect of nicotinic acid, which is an antioxidant, on the stability of PDSP in histidine-based buffers, 20 mM histidine containing 150 mM, 300 mM or 350 mM nicotinic acid was chosen. PDSP prepared in buffer (pH 7.0) for comparison. As shown in Figure 2 and Table 4 , for PDSP prepared in 20 mM histidine buffer containing 150-mM, 300-mM, and 350-mM nicotinamide, 5 and 4.5 seconds after starting stirring, respectively. and 14 hours suspended matter was observed ( Table 4 ). Suspended material formed significantly later in formulations containing histidine/nicotinamide buffer than in formulations containing citrate buffer.

In one study, the suspended material in PDSP prepared in 10 mM citrate buffer containing 0.85% NaCl was found to be coarse particles, and small particles or fibers could be observed under a dissecting microscope. However, the suspended material in the PDSP formulation prepared in histidine/nicotinamide buffer was extremely fine, which only reduced solution transparency without visible particles under a dissecting microscope.

To assess whether precipitation can also occur with the presence of suspended material, 400 µl aliquots of each PDSP solution were collected in 1.5 ml Eppendorf tubes and centrifuged (1,3000 rpm, 5 min). As shown in Figure 2 (middle panel) and Table 4 , for PDSP prepared in 20 mM histidine buffer containing 150-mM, 300-mM, and 350-mM nicotinamide, respectively, after starting stirring Visible precipitation was observed at 5.5 hours, 4.5 hours and 14.5 hours.

Once suspended material or precipitate is observed, continue stirring the PDSP solution until it becomes 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 reacted 13.5 hours, 13.5 hours after starting stirring, respectively. It becomes turbid between 7 and 24 hours and at 16.5 hours.

PDSP formulations prepared in histidine/nicotinamide buffer withstand shear stress better than PDSP formulations prepared in citrate buffer. Additionally, in these histidine/nicotinamide formulations, the solution containing 350 mM nicotinamide showed longer time to precipitate compared to the solutions containing 150 mM and 300 mM nicotinamide. , indicating that higher nicotinamide concentrations increase the stability of PDSP in formulations prepared in histidine-based buffers. 3. Resistance to shear force of PEDF-derived short peptides (PDSP) prepared in histidine/nicotinamide buffer containing different concentrations of sorbitol

Ocular administration of nicotinamide has been reported to cause eye irritation (Keri, G. 2005. Reassessment of the one experiment from the requirement of the tolerance for nicotinamide. United State Environmental Protection Agency Washington, DC 20460. 1-12). Therefore, sorbitol is used to replace all or part of the nicotinamide in histidine-based buffers. As shown in Table 4 , suspended material was observed just after 3 hours of stirring in the 20 mM histidine/260 mM sorbitol formulation alone. and in 20 mM histidine/150 mM nicotinamide containing 120-mM, 125-mM, 130-mM, 140-mM, 150-mM, 160-mM, 170-mM, and 180-mM sorbitol. In PDSP prepared in buffer, suspended substances were found at 3 hours, 4.5 hours, 4 hours, 13 hours, 13.5 hours, 6 hours, 5 hours and 4.5 hours after the start of stirring ( Figure 3 , Figure 4 and Table 4 ) .

Among these histidine/nicotinamide formulations, PDSP prepared in 20 mM histidine/150 mM nicotinamide containing 140-mM and 150-mM sorbitol was stirred continuously for 13 Suspended material, sedimentation, and turbidity were observed after hours, indicating that a range of approximately 140 mM to 150 mM may be the optimal concentration of sorbitol in the histidine/nicotinamide formulation. ( Figure 3 , Figure 4 and Table 4 ). Furthermore, the stability of PDSP prepared in 20 mM histidine/150 mM nicotinamide/140 or 150 mM sorbitol buffer and in only 20 mM histidine/350 mM nicotinamide buffer Quite, indicating that sorbitol can be used to replace a portion of nicotinamide.

These results, together with the data described above, indicate that formulations containing histidine/nicotinamide have better PDSP stability than citrate/NaCl formulations. For PDSP prepared in 10 mM citrate with 0.85% NaCl, precipitation occurred 1 hour after starting stirring, whereas for PDSP prepared in 20 mM histidine with 150 mM to 350 mM nicotinamide, Precipitation was observed after 5 hours of stirring. The 5-fold longer duration of precipitate formation in the Histidine/Nicotinamide formulation compared to the Citrate/NaCl formulation indicates that PDSP is significantly more stable in the Histidine/Nicotinamide formulation. In addition, among the formulations containing different concentrations of nicotinamide, precipitation was not observed until 14.5 hours after continuous stirring, indicating that higher concentrations of nicotinamide are more suitable as excipients to maintain the stability of PDSP.

PDSP formulations prepared in histidine/sorbitol only buffer showed a better ability to maintain PDSP stability compared to PDSP formulations prepared in citrate buffer. However, the ability to maintain PDSP stability was still not good enough for the histamine/sorbitol only formulation when compared to the histamine/nicotinamide only formulation, indicating that nicotinamide may be a useful tool for maintaining PDSP stability. Important component for the stability of PDSP in histidine-based buffers.

When a tonicity agent (such as sorbitol) is used to replace a portion of the nicotinamide, for PDSP prepared in 20 mM histidine/350 mM nicotinamide (14.5 hours) and in 20 mM histidine/150 mM Precipitation occurred at similar times for PDSP prepared in nicotinamide/140 or 150 mM sorbitol (13 and 14 hours, respectively). These data further confirm that a concentration of approximately 140 to 150 mM is a better choice for sorbitol concentration for PDSP formulations prepared in 20 mM histidine/150 mM nicotinamide buffer.

Overall, the formulations we tested indicate that histidine/nicotinamide is much better for formulations containing PDSP (such as PDSP; SEQ ID NO: 3) compared to citrate buffer Base buffer. According to embodiments of the present invention, the PDSP can be at any suitable concentration (such as 0.01% to 5% w/v, preferably 0.01% to 1% w/v) and the histidine buffer can be at any suitable concentration, such as 1 mM to 1% w/v. 100mM, preferably 5mM to 60mM, more preferably 10mM to 40mM, optimally 15mM to 30mM is used. The pH of the formulation may range from 5 to 9, preferably the pH is about neutral, such as 6.5 to 7.5, most preferably about 7.0. Such formulations contain an antioxidant, preferably nicotinamide, at a suitable concentration, such as 50 to 1000 mM, preferably 100 to 700 mM, more preferably 200 to 500 mM, and most preferably 300 to 400 mM. . For example, a preferred formulation of the PDSP solution may include 20 mM histidine and 350 mM nicotinamide, pH 7.0. Such formulations may also contain non-ionic tonicity agents at suitable concentrations, such as 0 to 500 mM, preferably 10 to 400 mM, more preferably 50 to 300 mM, and most preferably 100 to 200 mM. It is sorbitol. For example, a preferred formulation of the PDSP solution may include 20 mM histidine with 150 mM nicotinamide and 150 mM sorbitol, pH 7.0.

The formulations of the present invention can be used to treat various diseases and conditions, such as retinal degeneration, meibomian gland disease, dry eye, and the like. For ocular administration, the formulations may be ophthalmic solutions.

A limited number of examples have been used to illustrate embodiments of the invention. Those skilled in the art will understand that these examples are for illustration only and are not intended to limit the scope of the invention, as other modifications and changes may be made without departing from the scope of the invention. Therefore, the scope of the present invention should be limited by the appended claims.

Figure 1 shows a schematic diagram illustrating a testing protocol for assessing the stability of various formulations of PDSP solutions. Different PDSP solutions were prepared according to the study design. Adjust the pH value of the PDSP solution with 1N HCl or 2N NaOH, filter it through a 0.2 µm syringe filter, and put it into a 50 ml glass bottle. The filtered PDSP solution was stirred at room temperature at 1,150 RPM. Aliquots of 400 µl of the PDSP solution were collected at various time points (every half hour up to 7 or 9 hours) and centrifuged at 1,3000 rpm to observe if any precipitation occurred. The PDSP solution was continued to be stirred and precipitation was studied at the 10, 12, 18 and 24 hour time points. Record the time of appearance of suspended matter, sedimentation and turbidity.

Figure 2 shows the preparation under continuous stirring conditions in 10 mM citrate buffer (pH 6.0) containing 0.85% NaCl and in 20 mM histidine buffer (pH 7.0) containing various concentrations of nicotinamide. Stability test results of PDSP formulations. After filtration, each of the PDSP prepared in these different formulations was placed into a 50 mL beaker, and the solution was then stirred at 1,150 RPM at room temperature. The solutions were studied every half hour for the first 7 hours, and continuous observations were continued 12 hours after the start of stirring.

Figure 3 shows the results of stability testing of PDSP formulations prepared with different concentrations of sorbitol in a 20 mM histidine/150 mM nicotinamide solution. Stability testing was performed with continuous stirring. After filtration, PDSP prepared in 8 different formulations was placed into a 50 mL beaker, and the solution was then stirred at 1,150 RPM at room temperature. The solutions were studied every half hour for the first 9 hours after starting stirring and at 12, 18 and 24 hours. Record the time when precipitation and turbidity appear.

Figure 4 shows the time to onset of suspension, precipitation and turbidity in PDSP formulations prepared with varying concentrations of sorbitol in 20 mM histidine/150 mM nicotinamide solutions under continuous stirring conditions. Curve 1: The time when suspended matter appears. Curve 2: The time when precipitation appears. Curve 3: The time when the turbid solution appears.

         <![CDATA[<110>BRIM Biotechnology, Inc.]]>
          <![CDATA[<120> Compositions containing PEDF-derived short peptides (PDSP) and uses thereof]]>
          <![CDATA[<140>TW 109142968]]>
          <![CDATA[<141> 2020-12-04]]>
          <![CDATA[<150> ]]>US 62/911,367
          <![CDATA[<151> 2019-10-06]]>
          <![CDATA[<160> 9 ]]>
          <![CDATA[<170> PatentIn version 3.5]]>
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Claims (9)

An aqueous formulation comprising: PEDF-derivatized short peptide (PDSP) having the sequence of SEQ ID NO: 1, 2, 3, 5, 6, 8 or 9; nicotinamide having 50mM to 1000mM the concentration; and histidine, which has a concentration of 1mM to 100mM. The aqueous formulation of claim 1, wherein the pH value is about 5 to 9. The aqueous formulation of claim 1, further comprising a nonionic tonicity agent. The aqueous formulation of claim 3, wherein the nonionic tonicity agent is sorbitol. The aqueous formulation of claim 4, wherein the concentration of sorbitol is 0mM to 500mM. The aqueous formulation of claim 1, wherein the histidine concentration is 5mM to 60mM. The aqueous formulation of claim 1, wherein the histamine concentration is 10mM to 40mM. The aqueous formulation of any one of claims 1 to 7, wherein the PDSP has the sequence of SEQ ID NO: 3. For example, the aqueous formulation of claim 8, wherein the concentration of PDSP is 0.01% to 1% w/v.