Pharmaceutical composition for eardrum repair and use thereof

Abstract

A pharmaceutical composition for eardrum repair includes: collagen present in an amount of 8 wt % to 12 wt %; a forming agent present in an amount of 19 wt % to 22 wt %; and rest of a solvent, wherein the forming agent is a polymer of polyethylene oxide (PEO) and polypropylene oxide (PPO), polystyrene, polyethylene, polypropylene, polymethylmethacrylate, poly(N-isopropylacrylamide), poly[2-(dimethylamino)ethyl methacrylate] (pDMAEMA) hydroxypropylcellulose, poly(vinylcaprolactame), poly-2-isopropyl-2-oxazoline, polyvinyl methyl ether or a combination thereof.

Claims

1. A pharmaceutical composition for eardrum repair, comprising: collagen present in an amount of 8 wt % to 12 wt %; a forming agent present in an amount of 19 wt % to 22 wt %; and rest of a solvent, wherein the forming agent is a polymer of polyethylene oxide (PEO) and polypropylene oxide (PPO), polystyrene, polyethylene, polypropylene, polymethylmethacrylate, poly(N-isopropylacrylamide), poly[2-(dimethylamino)ethyl methacrylate] (pDMAEMA) hydroxypropylcellulose, poly(vinylcaprolactame), poly-2-isopropyl-2-oxazoline, polyvinyl methyl ether or a combination thereof.

2. The pharmaceutical composition of claim 1, wherein the forming agent is the polymer of PEO and PPO.

3. The pharmaceutical composition of claim 2, wherein the forming agent is a block copolymer of PEO and PPO.

4. The pharmaceutical composition of claim 3, wherein the forming agent is a PEO-PPO-PEO triblock copolymer.

5. The pharmaceutical composition of claim 2, wherein the block copolymer of PEO and PPO has a molecular weight ranging from 10,000 g/mol to 15,000 g/mol.

6. The pharmaceutical composition of claim 1, wherein the collagen is type I collagen.

7. The pharmaceutical composition of claim 6, wherein the collagen is type I reconstituted collagen.

8. The pharmaceutical composition of claim 1, which has a thermo-transition temperature ranging from 32° C. to 37° C.

9. The pharmaceutical composition of claim 1, which has a gelation time less than 120 seconds.

10. A method for repairing eardrum of a subject, comprising: administering to a subject in need thereof a pharmaceutical composition for eardrum repair, wherein the pharmaceutical composition comprises: collagen present in an amount of 8 wt % to 12 wt %; a forming agent present in an amount of 19 wt % to 22 wt %; and rest of a solvent, wherein the forming agent is a polymer of polyethylene oxide (PEO) and polypropylene oxide (PPO), polystyrene, polyethylene, polypropylene, polymethylmethacrylate, poly(N-isopropylacrylamide), poly[2-(dimethylamino)ethyl methacrylate] (pDMAEMA) hydroxypropylcellulose, poly(vinylcaprolactame), poly-2-isopropyl-2-oxazoline, polyvinyl methyl ether or a combination thereof.

11. The method of claim 10, wherein the forming agent is the polymer of PEO and PPO.

12. The method of claim 11, wherein the forming agent is a block copolymer of PEO and PPO.

13. The method of claim 12, wherein the forming agent is a PEO-PPO-PEO triblock copolymer.

14. The method of claim 11, wherein the block copolymer of PEO and PPO has a molecular weight ranging from 10,000 g/mol to 15,000 g/mol.

15. The method of claim 10, wherein the collagen is type I collagen.

16. The method of claim 15, wherein the collagen is type I reconstituted collagen.

17. The method of claim 10, wherein the pharmaceutical composition has a thermos-transition temperature ranging from 32° C. to 37° C.

18. The method of claim 10, wherein the pharmaceutical composition has a gelation time less than 120 seconds.

19. The method of claim 10, wherein the pharmaceutical composition is formulated into a formulation form of ear drops or sprays.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0035] FIG. 1 shows the results of gelation time test of the pharmaceutical composition of Example 1.

[0036] FIG. 2 shows the otoscope observation results before and after the surgery.

[0037] FIG. 3 shows the gross observation results of the eardrums of the experimental animals with or without the administration of the pharmaceutical composition of Example 1.

[0038] FIG. 4 shows the histopathological evaluation results of the eardrums having 50% defect of the experimental animals with or without the administration of the pharmaceutical composition of Example 1.

[0039] FIG. 5 shows the histopathological evaluation results of the eardrums having 75% defect of the experimental animals with or without the administration of the pharmaceutical composition of Example 1.

DETAILED DESCRIPTION OF EMBODIMENT

[0040] The following embodiments when read with the accompanying drawings are made to clearly exhibit the above-mentioned and other technical contents, features and/or effects of the present disclosure. Through the exposition by means of the specific embodiments, people would further understand the technical means and effects the present disclosure adopts to achieve the above-indicated objectives. Moreover, as the contents disclosed herein should be readily understood and can be implemented by a person skilled in the art, all equivalent changes or modifications which do not depart from the concept of the present disclosure should be encompassed by the appended claims.

Preparation of R-Collagen

[0041] Type I porcine collagen was purified by the method disclosed in Einbinder J. & Schubert M., J. Biol. Chem., 188,335(1951). The purified collagen was dissolved in ddH.sub.2O (10 mg/ml). The collagen solution was heated to 55° C. to 65° C., followed by adding HCl to obtain the collagen solution with pH 1.8˜2.2. Thus, the triple-helix of collagen can be denatured and dissolved evenly. Then, pepsin was added into the solution (in which the weight ratio of pepsin to collagen was 1:10) to remove the telopeptide at the N-terminal and the C-terminal of collagen, and the triple-helix of collagen can be converted into single α-chain collagen.

[0042] The obtained solution was treated with an alkaline solution until the pH of the solution was 10, to inhibit the activity of pepsin. Then, β-mercaptoethanol was added into the solution to reduce the remaining —S—S into —SH. Thus, the collagen can be completely converted into single α-chain collagen.

[0043] Then, glutaraldehyde (for example, 2%, 2.5% or 25%) was added into the collagen solution containing single α-chain collagen to perform the cross-linking process for 24 hours to 11 days. Glutaraldehyde can make asparagine, glutamine and arginine comprised in the collagen cross-linked to form glutaraldehyde-polymer amine complex, which is the reconstituted collagen (R-collagen).

[0044] The pH of the solution containing R-collagen was adjusted into neutral, followed by diluting with ddH.sub.2O (1:50, v/v), precipitated by centrifugation (30 min/10,000 rpm), and removing supernatant. The aforesaid steps were performed 8 to 10 times. The obtained R-collagen was not damaged, as well as the remaining glutaraldehyde can be removed.

[0045] The obtained R-collagen was dissolved in ddH.sub.2O (1:10, v/v), followed by slight oscillation to evenly dissolve the R-collagen. Then, the obtained solution was frozen at −70° C. for 8 hours or more, followed by lyophilization for 18˜24 hours to obtain the R-collagen powders.

Comparative Example 1

[0046] The pharmaceutical composition of the present comparative example was prepared as follows.

[0047] 3 g of the aforesaid R-collagen was dissolved in 3 ml of 5 mM AcOH. The collagen solution was heated to 40° C. for 4 hr with stirring, and the obtained R-collagen solution was stored at 4° C.

[0048] 5.4 g of Pluronic F127 (F127) (PEO98-PPO67-PEO98 triblock copolymer, CAS Number 9003-11-16) was dissolved in 18.6 ml ddH.sub.2O. The F127 solution was stirred on ice evenly, and the obtained F127 solution was stored at 4° C.

[0049] The obtained R-collagen solution was added into the obtained F127 solution at room temperature, and the mixture was stirred evenly until well mixed.

Example 1

[0050] The pharmaceutical composition of the present example was prepared by the same process illustrated in Comparative Example 1, except that 6.0 g of F127 was dissolved in 18.0 ml ddH.sub.2O.

Example 2

[0051] The pharmaceutical composition of the present example was prepared by the same process illustrated in Comparative Example 1, except that 6.6 g of F127 was dissolved in 17.4 ml ddH.sub.2O.

Comparative Example 2

[0052] The pharmaceutical composition of the present comparative example was prepared by dissolving 5.4 g of F127 in 18.6 ml ddH.sub.2O and stirring on ice evenly. The obtained F127 solution was stored at 4° C.

Comparative Example 3

[0053] The pharmaceutical composition of the present comparative example was prepared by the same process illustrated in Comparative example 2, except that 6.0 g of F127 was dissolved in 18.0 ml ddH.sub.2O.

[0054] The contents of the components in the obtained pharmaceutical compositions of Examples 1 and 2 and Comparative examples 1 to 3 are summarized in the following Table 1.

TABLE-US-00001 TABLE 1 Content Example Example Comparative Comparative Comparative Component 1 2 Example 1 Example 2 Example 3 F127 20 wt % 22 wt % 18 wt % 18 wt % 20 wt % R-collagen 10 wt % 10 wt % 10 wt % — —

Rheological Property Test

[0055] To determine viscosity changes of temperature versus various pharmaceutical compositions of Examples 1 and 2 and Comparative examples 1 to 3, the rheological property test was performed as follows.

[0056] 2 ml samples from Examples 1 and 2 and Comparative examples 1 to 3 were respectively loaded into viscometer channels. The temperatures of the samples were gradually increased from 4° C. to 36° C. (5° C./5 min), and the viscosity of each sample was recorded at 5 min interval. The results are listed in the following Table 2.

[0057] In Table 2, “F” refers to F127 and “C” refers to R-collagen, and tri-plicate experiments were performed on the groups of Examples 1 and 2 and Comparative Example 1.

TABLE-US-00002 TABLE 2 Temperature 4° C. 9° C. 14° C. 19° C. 24° C. 29° C. 34° C. 37° C. 39° C. 44° C. Example 1 142.9 285.7 1,143.3 1,342.0 6,286.0 10,714.0 11,857.0 11,571.0 11,571.0 11,429.0 20% F + 10% C Example 1 125.0 250.0 1000.0 1117.0 5625.0 9500.0 10500.0 10375.0 10375.0 10250.0 20% F + 10% C Example 1 100.0 200.0 900.0 1,000.0 4,500.0 7,800.0 8,900.0 8,600.0 8,600.0 8,500.0 20% F + 10% C Example 2 142.9 285.7 428.6 1,571.0 12,571.0 14,857.0 15,000.0 14,714.0 14,571.0 14,571.0 22% F + 10% C Example 2 125.0 250.0 500.0 1,375.0 11,375.0 13,375.0 13,500.0 13,125.0 13,000.0 12,875.0 22% F + 10% C Example 2 100.0 200.0 400.0 1,100.0 9,300.0 11,100.0 11,300.0 11,000.0 10,800.0 10,800.0 22% F + 10% C Comparative 142.9 142.9 142.9 142.9 142.9 285.7 285.7 285.7 142.9 142.9 Example 1 18% F + 10% C Comparative 125.0 125.0 125.0 125.0 125.0 250.0 250.0 250.0 125.0 125.0 Example 1 18% F + 10% C Comparative 100.0 100.0 100.0 100.0 100.0 200.0 200.0 200.0 100.0 100.0 Example 1 18% F + 10% C Comparative 83.3 83.3 83.3 83.3 166.7 2,583.0 4,167.0 4,417.0 — — Example 2 18% F-127 Comparative 20.0 20.0 20.0 2,000.0 6,000.0 8,000.0 9,000.0 10,000.0 10,000.0 — Example 3 20% F127

[0058] As shown in Table 2, the pharmaceutical composition comprising 20 wt % or 22 wt % F127 (Examples 1 and 2) showed a stable reverse thermal property versus temperature of 35° C.˜37° C. compared to the pharmaceutical composition comprising 18 wt % F127 (Comparative Example 1). This indicates a similar temperature of the ear canal.

[0059] In addition, compared to the pharmaceutical composition comprising 22 wt % F127 (Example 2), the pharmaceutical composition comprising 20 wt % F127 (Example 1) showed a relative stable rheological property and lower variation in viscosity change at 4° C.˜14° C.

[0060] Thus, the combination of 20 wt % F127 with 10 wt % collagen (Example 1) is the optimized pharmaceutical composition that shows stable rheological and reverse thermal gelation properties to achieve an optimal gelation at the ear canal temperature.

Gelation Time Test

[0061] The average time of gelation phase in the pharmaceutical composition comprising 20 wt % F127 and 10 wt % collagen (Example 1) was measured at the mimic temperature of the ear canal. The results are shown in FIG. 1, which indicates that the pharmaceutical composition of Example 1 has suitable gelation time.

Animal Model

Efficacy Test

[0062] Male SD rats were anesthetized with intramuscular ketamine hydrochloride (40 mg/kg) and xylazine hydrochloride (5 mg/kg) before surgery. The ears of all animals were assessed using an Olympus 3-mm otomicroscope before the procedure to rule out infection. Surgery of traumatic perforation of the tympanic membrane (TM) was performed under the guiding of otoscope with a 22 # intracatheter plastic tube needle passing through the ear canals anterior and posterior to the malleus handle, in the pars tensa region of the TM. 50% marginal perforation was created on anterior part, and 75% marginal perforation was created on anterior and half posterior part.

[0063] The experimental group is set as follows:

[0064] Eardrum defect group caused by surgery:

[0065] a1) 50% marginal perforation, n=12; and

[0066] b1) 75% marginal perforation, n=12.

[0067] Eardrum defect group caused by surgery and administered with 0.3-0.5 ml of the pharmaceutical composition of Example 1:

[0068] a2) 50% marginal perforation, n=12; and

[0069] b2) 75% marginal perforation, n=12.

[0070] Half operations were performed on left side, and the other half were on the right to avoid the confounder effect.

[0071] The test endpoint is set as follows.

[0072] After the surgery, the wound healing of the eardrum was observed and recorded by using an otoscope, wherein the images of the suppuration and inflammation of the ear canal and inner ear were recorded, and the health statuses of the experimental animals were also evaluated. The observation was performed once every 3 days, 10 times, for a total of 30 days. The animals were sacrificed after the 10th observation.

[0073] After the animals were sacrificed, the eardrum tissue was excised en bloc for histological analysis. The repair of the eardrum epithelial tissue was observed, and the immune cell infiltration status of the inner ear tissue was evaluated the degree of the inflammation and suppuration.

[0074] The results of the otoscope observation before and after the surgery are shown in FIG. 2, and the results of the gross observation are shown in FIG. 3. In FIG. 3, “Post” refers to the posterior part, and “Ant” refers to the anterior part. The results indicate that the eardrum defects (50% and 75% defects) can be effectively repaired when the pharmaceutical composition of Example 1 was administered to the experimental animals.

[0075] The results of the histopathological evaluation are shown in FIG. 4 and FIG. 5.

[0076] As shown in FIG. 4, under histological evaluation, the group with the administration of the pharmaceutical composition of Example 1 showed an ingrowth and recovery of the tympanic membrane tissue at the 50% defect site. In the control group without the administration of the pharmaceutical composition of Example 1, a rupture membrane structure was observed, with severe immune cell infiltration and inflammation at the adjacent tissue.

[0077] As shown in FIG. 5, the group with the administration of the pharmaceutical composition of Example 1 showed a decent membrane regeneration effect, while an intact tympanic membrane was observed without signs of inflammation in the adjacent tissue. In the control group without the administration of the pharmaceutical composition of Example 1, perforated tissue with sever fibrosis was observed at the defect site.

[0078] These results implied that the pharmaceutical composition of the present disclosure could provide a suitable tissue matrix that supports the epithelial and fibroblast cell ingrowth along the direction of the membrane thus regenerates the tympanic membrane tissue, even in the large defect case.

[0079] Although the present disclosure has been explained in relation to its embodiment, it is to be understood that many other possible modifications and variations can be made without departing from the spirit and scope of the disclosure as hereinafter claimed.