NOVEL SUSTAINED RELEASE COMPOSITION FOR WOUND HEALING FIELD OF THE INVENTION

The present invention relates to novel composition for wound healing, that is incorporated into a medical dressing having sustained delivery of the drug and process for the preparation of the same.

BACKGROUND OF THE INVENTION

Successful wound healing depends upon angiogenesis, the growth of new capillary blood vessels. Wound fluid stimulates vascular endothelial cells to migrate and proliferate in vitro and induces angiogenesis in vivo. The angiogenic process involves growth factor activation of endothelial cells, leading to proliferation, migration, tubular morphogenesis, vascular loop formation, and stabilization of vessels to form a mature vascular network.

Wound healing occurs in three major overlapping stages, a haemostatic and inflammatory stage, a proliferative stage and remodeling stage. Although granulation is classically assigned to the proliferative stage, angiogenesis is initiated immediately upon wounding and is mediated throughout the entire wound-healing process which promotes the local dissolution of basement membrane, an essential early step of angiogenesis. Recent advances in wound biology have led to important insights on how growth factors mediate these processes. Several growth factors (PDGF, VEGF, and bFGF) synergize in their ability to vascularize tissues.
PDGF plays an important role in the vascular stabilization in the wound healing process. Deficiency in the PDGF leads to abnormal, poorly formed; immature blood vessels. Diabetes is associated with a reduction in the expression of growth factors and their receptors. Compared with non-diabetic people, levels of PDGF in diabetic wounds are decreased in the early phases of healing. Therefore PDGF is considered to be very essential in the wound healing process. PDGF-BB is a potent stimulator of angiogenesis that also stabilizes newly formed blood vessels. I Keeping in view, the therapeutic benefits of PDGF-BB the protein was produced in large scales, using recombinant DNA technology. PDGF-BB is now available as gel formulation which needs to be applied to the wound once a day as the protein is released on the wound at once. Because of the low half life of the protein in the body the number of dosages of the protein are more and uncontrolled. Moreover the gel based formulation is stable only at 2-8°C. With the present invention we attempt to overcome these challenges.

The present invention therefore relates to a novel, storage stable composition and sustained release formulation for PDGF-BB that eliminates necessity of daily wound dressing and improves the stability of protein at 25°C.

OBJECTIVE OF THE INVENTION

An object of the invention is to provide a novel composition of PDGF-BB that may be incorporated into a matrix so as to produce a sustained release.

SUMMARY OF THE INVENTION

The present invention relates to a novel, storage stable, synergistic composition of PDGF-BB. The novel composition comprises of PDGF-BB in conjunction with carbohydrates and a cross linking agent. The composition of the present invention is stable for extended periods over significant range of temperatures, without producing any degradants.

BRIEF DESCRIPTION OF FIGURES

Fig 1: SDS-PAGE analysis of PDGF-BB released from the Medical dressing of the composition of example 1

Fig: 2 PDGF-BB concentration in the released samples from the Medical dressing of the composition of example 1

Fig 3: PDGF-BB Activity (Bioassay) in the released samples from the Medical dressing of the composition of example 1

Fig 4: SDS-PAGE analysis of PDGF-BB released from the Medical dressing of the composition of example 2

Fig: 5 PDGF-BB concentration in the released samples from the Medical dressing of the composition of example 2

Fig 6: PDGF-BB Activity (Bioassay) in the released samples from the Medical dressing of the composition of example 2

Fig 7: SDS-PAGE analysis of PDGF-BB released from the Medical dressing of the composition of example 3

Fig: 8 PDGF-BB concentration in the released samples from the Medical dressing of the composition of example 3

Fig 9: PDGF-BB Activity (Bioassay) in the released samples from the Medical dressing of the composition of example 3

Fig 10: SDS-PAGE analysis of PDGF-BB released from the Medical dressing of the composition of example 4

Fig 11: PDGF-BB concentration in the released samples from the Medical dressing of the composition of example 4

Fig 12: PDGF-BB Activity (Bioassay) in the released samples from the Medical dressing of the composition of example 4

Fig 13: SDS-PAGE analysis of PDGF-BB released from the Medical dressing of the composition of example 5

Fig 14: PDGF-BB concentration in the released samples from the Medical dressing of the composition of example 5

Fig IS: PDGF-BB Activity (Bioassay) in the released samples from the Medical dressing of the composition of example 5

Fig 16: SDS-PAGE analysis of PDGF-BB released from the Medical dressing of the composition of example 6

Fig 17: PDGF-BB concentration in the released samples from the Medical dressing of the composition of example 6

Fig 18: PDGF-BB Activity (Bioassay) in the released samples from the Medical dressing of the composition of example 6

DETAILED DESCRIPTION OF THE INVENTION:

Accordingly present invention provides a novel, sustained release, storage stable and synergistic composition of PDGF-BB. All terms used herein, carry the same meaning as used in common scientific parlance.

The composition of the present invention comprises PDGF-BB, a carbohydrate and a cross linking agent.

The PDGF-BB used in the composition may be derived from any source such as natural source, synthetic source or recombinant source. Preferably, PDGF-BB as used in the composition is produced by recombinant technology. For instance, PDGF-BB may be produced from E. colt.
The amount of PDGF-BB present in the composition may be in the range of 20microgram to 60microgram/cm2. Most preferably, the amount of PDGF-BB is in the range of 30microgram to 50 microgram/cm2. The pH of the composition is maintained in the range of 4.0 to 6.0.
The carbohydrate used in the present invention is selected from the group comprising ionic polysaccharides such as uronic acids, sulphated polysaccharides or muco
polysaccharides or their mixtures thereof. Some non limiting examples of polysaccharides include hyaluronic acid, carboxymethyl cellulose (CMC), chondroitin -6- sulphate and heparin sulphate. The carbohydrate used is preferably a mixture of hyaluronic acid and carboxy methyl cellulose.

The total carbohydrate present in the composition may be in the range of 0.1 to 1.0%. The concentration of the individual components such as hyaluronic acid may be in the range of 0.1 to 1.0%, preferably 0.5 to 0.8% and the concentration of carboxy methyl cellulose may be in the range of 0.1 to 0.8% preferably 0.2 to 0.5%.

The cross linking agent of the present invention is selected from the group comprising carbodiimides, bis carbodiimides, epoxides, aldehydes, etc. Preferably the cross linking agent is selected from carbodiimides. The amount of cross linking agent present in composition is in the range of 0.5 to 2.0%, preferably 0.8 to 1.2%.

In yet another aspect of the invention, the composition of the present invention, may be prepared by a process comprising the steps of

i. Mixing the cross linking agent and the carbohydrate

ii. Drying the cross linked material and suspending the same.

iii. Mixing the PDGF-BB protein to the suspension of step ii and drying the composition.

The mixing of the cross linking agent may be carried out by stirring or, homogenization or by any other process, which renders fine blending of the ingredients. The drying of the mixture as disclosed in step (ii) may be carried out by any technique such as vacuum drying, lyophilizing, or air drying preferably the drying is carried out by lyophilizing.

In another embodiment the process of lyophilization involves a drying step may be carried out in one or two steps. The time taken for the whole process may range from 9 - 10 hours. The drying may be performed for 9 hours with a gradual increase of temperature from -15°C to +25°C and vacuum maintained at 200mT. The process of lyophilization also involves a drying step. The time taken for the whole process may range from 9-10 hrs. The drying steps may be performed for 9-10 hrs with a gradual increase of temperature from -10°C to +30°C and pressure is varied from 180 to 220mT. The drying may be done in several cycles and the final unloading of the lyophilized product may be performed at room temperature and ambient pressure.

Without being limited by theory, it is submitted that novel composition of the present invention is such that the PDGF-BB maintains stability during the various steps involved in the preparation of the dressing such as mixing, drying and protein extraction from the dressing. Further, the formulation of the present invention withstands various physical constraints and results in a rigid medical dressing. Furthermore, the formulation of the present invention is stable for extended periods of time at elevated temperature. In particular, the formulation of the present invention maintains the chemical and biological stability at higher temperatures (25°C). It is proposed that the optimum use of the various ingredients at the said ratio maintains the stability of the formulation.

Further, the composition of the present invention contains a pure grade of Platelet derived growth factor-BB which is greater than 95.0%. With this higher % purity the formulation is stabilized welt.

In another aspect, the composition of the present invention is used for wound healing. Further, the wound healing effect is sustained, thereby eliminating the need for frequent changing of dressings.

The following examples are provided herein as a means of illustration but are not meant in any way to restrict the effective scope of the invention.

Example 1: Process for Preparation of composition

The novel composition of the present invention is prepared by dissolving 600mg of sodium hyaluronate and 400mg of sodium carboxy methyl cellulose in 75ml of water by stirring O/N on a magnetic stirrer (solution A) and solution-B is prepared freshly by dissolving lg of EDC-HCI (1 Ethyl 3, 3 dimethylaminopropyl carbidiimide-HCI) in 25ml of water. Solutions A and B are mixed and stirred on a magnetic stirrer for 30 minutes (till uniform mixing). Adjust the pH of the mixed solution between 4.0 to 5.0 with 0.1N HCI. To this mixture, 8ml of 3M NaCI is added which acts as an inducing agent for the precipitation of cross linked sodium hyaluronate and sodium carboxy methyl cellulose. Then iso propyl alcohol (IPA) is added to this mixture in the ratio 1:2 (1 of the HA, CMC cross linked mixture and 2 parts of IPA) and mixed gently and incubated at 25°C for 1 hour. The liquid is removed and the precipitate is collected. The collected precipitate is washed twice with each of 70%, 90% and 100% IPA to remove the residual EDC and sodium chloride. The precipitate is lyophilized for the removal of residual IPA at -15°C for 60minutes, -5°C for 60minutes, +5°C for 60minutes, +15°C for 120minutes and +25°C for 60minutes.

500mg of the lyophilized material is dissolved in 50ml of water and incubated at 60°C for 18 hours for hydration. The hydrated gel is homogenized at 17000rpm, for 20 minutes and incubated in a water bath, maintained at 98°C for 2 hours (to make a uniform suspension). The suspension is cooled to room temperature and then PDGF-BB is added to this solution at a concentration of 1.0 to 3.0 mg/ml more preferably 1.5 to 2.5mg/ml such that the final concentration in the suspension is 40 microgram/ml (2mg/50ml). The suspension is poured in an acrylic tray (12cmx8.5cm to a height of 0.5cm) to provide the composition of the present invention and placed in an oven at 40°C for 2 days to prepare the dressing.
Release profile of PDGF-BB of the composition as prepared by Example 1

The composition as prepared by the process as discussed in example 1 is taken and 7.5cm2 film is soaked in normal saline and left for several hours. The saline is changed at different hours (3, 6, 24, 48 and 72 hours) and the PDGF-BB quantities are assessed by protein estimation and analyzed for biological activity by cell proliferative assay. At three hours the PDGF-BB released could be due to the proteins located on the surface of the films. Subsequent hours, up to 72 hours, the release of PDGF-BB is observed. The cell proliferation activity per mg of PDGF-BB remains the same indicating that the activity is not altered by preparation of films wherein PDGF-BB is incorporated. The molecular weight of the released PDGF-BB is confirmed by SDS-PAGE which shows the expected size of about 30kDa indicating the process of making PDGF-BB films did not alter its molecular size. For example, it can be seen from the Figures of SDS-PAGE that the protein is released for 72 hours. From Fig. 1, it may be observed that the composition of example 1 is released for 72 hours. From Fig 2, it may be inferred that the concentration of PDGF-BB released is adequate for obtaining a therapeutic benefit for 72 hours and from Fig 3. It may be inferred that the composition of the present invention retains its biological activity for 72 hours.

Example 2:

Example 2, lists the quantity of the components and parameters for preparation of the composition of the present invention.

Components: QUANTITIES

1. Sodium hyaluronate 700 mg
2. Carboxymethyl cellulose 300 mg
3. EDC.HCI 1000 mg
4. pH 4.0-5.0
above ingredients are dissolved in 100ml of water and precipitated and lyophilized as described in example 1. 500mg of the precipitate is taken and added to 50ml of water.
5. Hydration temperature 60°C
6. Hydration time 18 Hrs
7. Incubation (after homogenization) temperature 98°C
8. Incubation (after homogenization) time 2 Hrs
9. PDGF-BB added 2mg (4mg/100ml)
10. Drying temperature 40°C
11. Drying time 2 days

The release profile of the composition as prepared in example 2 is listed in Fig 4, 5 and Fig 6. From Fig. 4, it may be observed that the composition of example 1 is released for 72 hours. From Fig 5, it may be inferred that the concentration of PDGF-BB released is adequate for obtaining a therapeutic benefit for 72 hours and from Fig 6. It may be inferred that the composition of the present invention retains its biological activity for 72 hours.

Example 3: Process for Preparation of composition

Example 3, lists the quantity of the components and parameters for preparation of the composition of the present invention.

COMPONENTS QUANTITIES

1. Sodium hyaluronate 500 mg
2. Carboxymethyl cellulose 500 mg
3. EDC.HCI 800 mg
4. pH 4.5-5.0
above ingredients are dissolved in 100ml of water and precipitated and lyophilized as described above. 500mg of the precipitate is taken and added to 50ml of water.
5. Hydration temperature 60°C
6. Hydration time 18 Hrs
7. Incubation (after homogenization) temperature 98°C
8. Incubation (after homogenization) time 2 Hrs
9. PDGF-BB added 4mg (8mg/100ml)
10. Drying temperature 40°C
11. Drying time 2 days

The release profile of the composition as prepared in example 3 is listed in Fig 7, 8 and Fig 9. From Fig. 7, it may be observed that the composition of example 1 is released for 72 hours. From Fig k8, it may be inferred that the concentration of PDGF-BB released is adequate for obtaining a therapeutic benefit for 72 hours and from Fig 9. It may be inferred that the composition of the present invention retains its biological activity for 72 hours.

Example 4: Process for Preparation of composition

Example 4, lists the quantity of the components and parameters for preparation of the composition of the present invention.

COMPONENTS QUANTITIES

1. Sodium hyaluronate 800 mg
2. Carboxymethyl cellulose 200 mg
3. EDC.HCI 1100 mg
4. pH 4.5-5.5
above ingredients are dissolved in 100ml of water and precipitated and lyophilized as described above. 500mg of the precipitate is taken and added to 50ml of water.
5. Hydration temperature 60°C
6. Hydration time 18Hrs
7. Incubation (after homogenization) temperature 98°C
8. Incubation (after homogenization) time 2 Hrs
9. PDGF-BB added 5mg (10mg/100ml)
10. Drying temperature 40°C
11. Drying time 2 days

The release profile of the composition as prepared in example 3 is listed in Fig 10, 11 and Fig 12. From Fig. 10, it may be observed that the composition of example 4 is released for 72 hours. From Fig 11, it may be inferred that the concentration of PDGF-BB released is adequate for obtaining a therapeutic benefit for 72 hours and from Fig 12. It may be inferred that the composition of the present invention retains its biological activity for 72 hours.

Example 5: Process for Preparation of composition

Example 5, lists the quantity of the components and parameters for preparation of the composition of the present invention.

COMPONENTS QUANTITIES

1. Sodium hyaluronate 700 mg
2. Carboxymethyl cellulose 300 mg
3. EDC.HCI 900 mg
4. pH 4.5-5.5
above ingredients are dissolved in 100ml of water and precipitated and lyophilized as described above. 500mg of the precipitate is taken and added to 50ml of water.
5. Hydration temperature 50°C
6. Hydration time 48 Hrs
7. Incubation (after homogenization) temperature 98°C
8. Incubation (after homogenization) time 2 Hrs
9. PDGF-BB added 6mg (12mg/100ml)
10. Drying temperature 40°C
11. Drying time 2 days

The release profile of the composition as prepared in example 5 is listed in Fig 13, 14 and Fig 15. From Fig. 13, it may be observed that the composition of example 5 is released for 72 hours. From Fig 14, it may be inferred that the concentration of PDGF-BB released is adequate for obtaining a therapeutic benefit for 72 hours and from Fig 15. It
may be inferred that the composition of the present invention retains its biological activity for 72 hours.

Example 6: Process for Preparation of composition

Example 6, lists the quantity of the components and parameters for preparation of the composition of the present invention.

COMPONENTS: QUANTITIES

1. Sodium hyaluronate 500 mg
2. Carboxymethyl cellulose 500 mg
3. EDC.HCI 1000 mg
4. pH 4.5-5.0
above ingredients are dissolved in 100ml of water and precipitated and lyophilized as described above. 500mg of the precipitate is taken and added to 50ml of water.
5. Hydration temperature 60°C
6. Hydration time 24Hrs
7. Incubation (after homogenization) temperature 98°C
8. Incubation (after homogenization) time 2 Hrs
9. PDGF-BB added 6mg (12mg/100ml)
10. Drying temperature 40°C
11. Drying time 2 days

The release profile of the composition as prepared in example 6 is listed in Fig 16, 17 and Fig 18. From Fig. 16, it may be observed that the composition of example 6 is released for 72 hours. From Fig 17, it may be inferred that the concentration of PDGF-BB released is adequate for obtaining a therapeutic benefit for 72 hours and from Fig 18. It may be inferred that the composition of the present invention retains its biological activity for 72 hours.
As mentioned in the examples, wound dressings with different compositions of hyaluronic acid, carboxy methyl cellulose and PDGF-BB are prepared and studied. The release of the protein is observed to be maximum at 24 hour and 48 hour durations. The biological activity of the protein is assayed and it is observed that protein released from all the dressings, showed a similar activity which indicates that the quality of protein is not altered because of the formulation.

ADVANTAGE OF THE INVENTION:

1. The novel composition of the present invention is synergistic and has enhanced storage stability

2. The composition of the present invention is stable for a period of 2 years at a temperature of 2°C to 8°C and upto 1 year at 25°C

3. The composition of the present invention retains the biological activity for a period of 2 years when stored at a temperature of 2°C to 8°C and up to 1 year at 25°C.

CLAIMS

We claim:

1. A novel, synergistic composition of PDGF-BB comprising a carbohydrate and a cross linking agent.

2. A novel composition of PDGF-BB as claimed in claim 1, wherein the PDGF-BB is obtained from natural source, synthetic source or recombinant source.

3. A novel composition of PDGF-BB as claimed in claim 1, wherein the PDGF-BB is preferably from recombinant source.

4. A novel composition of PDGF-BB as claimed in claim 1, wherein the amount Gf PDGF-BB is 20microgram to 60microgram/cm2, preferably, 30microgram to 50 microgram/cm2.

5. A novel composition of PDGF-BB as claimed in claim 1, wherein the carbohydrate is selected from the group comprising ionic polysaccharides such as uronic acids, sulphated polysaccharides or muco polysaccharides or their mixtures thereof.

6. A novel composition of PDGF-BB as claimed in claim 1, wherein the carbohydrate is a mixture of hyaluronic acid, carboxymethyl cellulose (CMC), chondroitin -6- sulphate and heparin sulphate.

7. A novel composition of PDGF-BB as claimed in claim 1, wherein, the total carbohydrate present is in the range of 0.1 to 1.0%.

8. A novel composition of PDGF-BB as claimed in claim 1, wherein, the concentration hyaluronic acid is the range of 0.1 to 1.0%, preferably 0.5 to 0.8% and the concentration of carboxy methyl cellulose is in the range of 0.1 to 0.8% preferably 0.2 to 0.5%.

9. A novel composition of PDGF-BB as claimed in claim 1, wherein the cross linking agent is selected from the group comprising carbodiimides, bis carbodiimides, epoxides, aldehydes, preferably carbodiimides.

10. A novel composition of PDGF-BB as claimed in claim 1, wherein the crosslinking agent is in the range of 0.5 to 2.0%, preferably 0.8 to 1.2%.

11. A novel composition of PDGF-BB as claimed in claim 1, prepared by a process comprising the steps of:

i. Mixing the cross linking agent and the carbohydrate
ii. Drying the cross linked material and suspending the same.
iii. Mixing the PDGF-BB protein to the suspension of step and drying the composition.

12. A process for preparation of the composition as claimed in claim 10, wherein the mixing in step (i) and (iii) of the cross linking agent is by stirring or, homogenization or by any other process and drying is by vacuum drying, lyophylization or air drying, preferably lyophylization.

13. A process for preparation of the composition as claimed in claim 11, wherein the lyophilization is carried for 9 -10 hours, with a gradual increase of temperature from-10°C to+30°C and vacuum maintained at 180 to 220mT.

14. A process for preparation of the composition as claimed in claim 11, wherein the process of lyophilization is done in several cycles and the final unloading of the lyophilized product is at room temperature and ambient pressure.

15. A novel composition as claimed in claim 1 for use in healing of woupds