Composition containing hyaluronidase

Abstract

Disclosed are microneedles comprising a hyaluronidase.

Description

Composition containing hyaluronidase

[0001] The present invention relates to a composition containing a peptide agent. More specifically, the present invention relates to a composition for treating inflammation, comprising a peptide (particularly, hyaluronidase) as an active ingredient, and for coating microneedles.

[0002] A drug delivery device including microneedles is a transdermal drug delivery system that allows microneedles with a length of approximately several hundred micrometers to pass through the stratum corneum of the skin and deliver active ingredients into the skin.

[0003] Microneedles are expected to efficiently achieve transdermal delivery of active substances while minimizing pain caused by skin invasion by combining the transdermal delivery efficacy of existing injectable formulations with the convenience of patches, a transdermal drug delivery system.

[0004] However, in reality, there have been no cases of microneedle formulations being developed and commercialized as pharmaceuticals, and they are only being commercialized for skin care purposes such as cosmetics.

[0005] In particular, when biopharmaceuticals containing biochemical substances, such as peptides, as active ingredients are administered orally, their bioavailability is very low because the active biochemical substances are degraded in the gastrointestinal tract. Consequently, most biopharmaceuticals are formulated and administered as injections. However, injections are painful and require specialized medical personnel. Furthermore, their large molecular weight makes transdermal administration difficult.

[0006] The present invention provides a formulation that can be administered transdermally, changing the route of administration of biopharmaceuticals, which were previously administered by injection. Specifically, the present invention provides a drug delivery device containing microneedles coated with a drug layer containing hyaluronidase. Furthermore, the present invention provides a composition for treating inflammation containing a peptide (particularly, hyaluronidase) for coating at least a portion of the microneedles.

[0007] Microneedle formulations have the advantage of being unaffected by the molecular weight of the active ingredient, causing less pain, and allowing self-administration, as the microneedles themselves pass directly through the stratum corneum to deliver the active ingredient into the body.

[0008] The present invention relates to a composition for coating at least a portion of the surface of a microneedle in a microneedle formulation for transdermal drug administration, the composition comprising hyaluronidase and a hydrophilic polymer as active ingredients. The composition of the present invention is effective in suppressing edema or treating inflammation. Therefore, the composition of the present invention and / or a drug delivery device containing a microneedle coated with the composition of the present invention can be used for treating edema or inflammation.

[0009] In the composition of the present invention, it is preferable that hyaluronidase is contained in an amount of 20% or less relative to the total weight of the composition.

[0010] In the composition of the present invention, the average molecular weight of the hydrophilic polymer material is preferably 300 to 1300 kDa. As the hydrophilic polymer material, at least one selected from the group consisting of HPMC (hydroxypropylmethyl cellulose), HPC (hydroxypropyl cellulose), HEC (hydroxyethyl cellulose), PVP (polyvinylpyrrolidone), PVA (polyvinyl alcohol), and carboxymethyl cellulose may be used. Preferably, HEC, PVP, or a mixture thereof may be used as the hydrophilic polymer material.

[0011] The composition of the present invention may further include a surfactant. For example, the composition of the present invention may include at least one surfactant selected from the group consisting of poloxamer, sorbitan ester (span), and polysorbate (tween).

[0012] In addition, the present invention relates to a drug delivery device for suppressing edema or treating inflammation, comprising a microneedle; and a support for supporting the microneedle, wherein at least a portion of the surface of the microneedle is coated with a drug layer containing hyaluronidase as an active ingredient and a hydrophilic polymer.

[0013] In the drug delivery device of the present invention, the drug layer may further include a surfactant (e.g., poloxamer, sorbitan ester (span), polysorbate (tween), etc.).

[0014] In the drug delivery device of the present invention, the area of ​​the support (particularly, the area of ​​the portion where the microneedles are formed) is about 1 to 25 cm 2 It may be, but is not limited to this range.

[0015] The hydrophilic polymer material used in the drug delivery device of the present invention may have an average molecular weight of 300 to 1300 kDa. As the hydrophilic polymer material, at least one selected from the group consisting of HPMC (hydroxypropylmethyl cellulose), HPC (hydroxypropyl cellulose), HEC (hydroxyethyl cellulose), PVP (polyvinylpyrrolidone), PVA (polyvinyl alcohol), and carboxymethyl cellulose may be used. Preferably, HEC, PVP, or a mixture thereof may be used as the hydrophilic polymer material.

[0016] The composition according to the present invention can be manufactured by adding an additive such as a hydrophilic polymer and purified water to the main ingredient raw material (hyaluronidase) and stirring.

[0017] The above additives include hydrophilic polymers with swelling properties, such as hydroxyethyl cellulose, hydroxypropylmethyl cellulose, hydroxypropyl cellulose, polyvinylpyrrolidone, polyvinyl alcohol, and carboxymethyl cellulose. If necessary, stabilizers and / or surfactants may be additionally added as additives.

[0018] Hyaluronidase-coated microneedles can be manufactured by immersing the microneedles in the above-mentioned composition and then drying them.

[0019] When the above microneedles are inserted into the body, the polymer swells, allowing the drug to be gradually released. Therefore, the drug release rate can be controlled to achieve desired pharmacokinetic properties.

[0020] In the drug delivery device of the present invention, the microneedle and the support supporting the microneedle may be made of any material that can be formed into a microneedle, for example, silicon, metal, ceramic, polymer, etc.

[0021] In the drug delivery device of the present invention, the length of the microneedle may be about 100 to 800 μm, and the aspect ratio (i.e., the ratio of the length of the microneedle to the maximum diameter of the microneedle) may be about 1:2 to 1:10, but is not limited to this range.

[0022] The support of the drug delivery device according to the present invention can be manufactured in any size as needed, and even if manufactured in a small size, an amount of drug sufficient to obtain the desired pharmacological effect can penetrate the skin.

[0023] Hyaluronidase, the main ingredient that exhibits the efficacy of the present invention, has a large molecular weight and a very short half-life in plasma.

[0024] However, the composition and drug delivery device according to the present invention can have a high skin permeation rate and drug delivery rate of the drug, and can also allow the effect of the drug to be continuously shown for a longer period of time.

[0025] Figure 1 schematically illustrates a cross-section of a manufactured microneedle.

[0026] Figure 2 is a photograph of a microneedle manufactured by coating twice with a coating solution using PVP (A), PVA (B), and HEC (C), respectively.

[0027] Figure 3 is a photograph taken under a microscope of the state of the coating solution over time during a dissolution rate test of the drug delivery device according to the present invention.

[0028] Figure 4 is a photograph showing the gelation state when a coating solution was prepared using HPMC and HPC.

[0029] Figure 5 shows the results of measuring the shear viscosity of a coating solution using a rheometer.

[0030] Figure 6 is a graph showing the cumulative dissolution rate of the dissolution test results of the drug delivery device according to the present invention.

[0031] Figure 7 is a graph showing the cumulative dissolution rate when the hydrophilic polymers HEC and PVP were used alone and when they were used in combination.

[0032] Hereinafter, to aid understanding of the present invention, examples will be provided in detail. However, the following examples merely illustrate the scope of the present invention and are not intended to limit its scope. These examples are provided to more fully explain the present invention to those of average skill in the art.

[0033] Example 1: Manufacturing of microneedles using a coating method

[0034] Example 1-1: Preparation of coating solution to be used as coating layer

[0035] Example 1-1-1: Coating solution composed of main ingredient and hydrophilic polymer

[0036] As a thickener (hydrophilic polymer), one of hydroxyethyl cellulose (HEC), hydroxypropyl methyl cellulose (HPMC), hydroxypropyl cellulose (HPC), polyvinylpyrrolidone (PVP), and polyvinyl alcohol (PVA) was selected, and added to purified water to concentrations of 3, 3, 20, 20, and 30 w / w%, respectively, and stirred using a roll mixer for 24 hours.

[0037] Thereafter, the main ingredient (hyaluronidase) was added to the prepared mixed solution at a concentration of 10 to 20 w / w% and stirred for 1 hour. The concentrations of HEC, HPMC, HPC, PVP, and PVA in the coating solution were set to 2.5, 2, 10, 18, and 26 w / w%, respectively.

[0038] Example 1-1-2: Coating solution composed of main ingredient, hydrophilic polymer, and surfactant

[0039] As a thickener (hydrophilic polymer), select one of hydroxyethyl cellulose (HEC), polyvinylpyrrolidone (PVP), and polyvinyl alcohol (PVA), and add them to purified water at concentrations of 3, 20, and 30 w / w%, respectively.

[0040] Poloxamer (Lutrol) as a surfactant ® ) was added to the above solution to a concentration of 1 w / w% and stirred for 24 hours using a roll mixer.

[0041] Thereafter, the main ingredient (hyaluronidase) was added to the prepared mixed solution at a concentration of 10 to 20 w / w% and stirred for 1 hour. The concentrations of HEC, PVP, and PVA in the coating solution were set to 2.5, 18, and 26 w / w%, respectively.

[0042] The coating solution composition of Example 1-1-1 and the coating solution composition of Example 1-1-2 are shown in Tables 1 and 2 below, respectively.

[0043] Coating solutionMain ingredient (w / w%)Thickener (w / w%)Purified water (w / w%)HEC16.72.580.8HPMC12.52.085.5HPC14.010.076.0PVP14.518.067.5PVA13.326.060.7

[0044]

[0045] Coating solutionMain ingredient (w / w%)Thickener (w / w%)Surfactant (w / w%)Purified water (w / w%)HEC16.52.51.080.1PVP14.318.01.066.7PVA13.225.71.060.1

[0046]

[0047] Example 1-2: Preparation of support

[0048] A total of 338 microneedles measuring 450 μm in length and 150 μm in width were designed to be distributed within a circle measuring 1 cm in diameter. A negative PDMS (polydimethylsiloxane) mold was fabricated using a positive metal mold manufactured according to this design. PLA (polylactic acid) pellets were placed inside the PDMS mold manufactured in this manner and heated in an oven at 195°C for 1 hour to melt them. Afterwards, the mold was taken out of the oven and completely cooled at room temperature. The solidified microneedles and support made of PLA material were separated from the completely cooled PDMS negative mold, and a support in which the microneedles were arranged in an integral form was manufactured. Figure 1 schematically illustrates a cross-section of the microneedles manufactured in this manner.

[0049] Example 1-3: Microneedle coating

[0050] Using a dip coating method, the surface of the microneedle support was coated with the coating solution prepared in Example 1-1.

[0051] Specifically, using a dip coating device, PLA microneedles were vacuum-absorbed, and the coating solution was applied into a coating solution reservoir with a diameter of 1.5 cm and a depth of 300 μm. The PLA microneedles, adsorbed and fixed in the vacuum absorber, were lowered at a constant speed to the reservoir onto which the coating solution was applied, thereby immersing the microneedles in the coating solution and coating them. This coating process was repeated twice. Afterwards, the samples were dried in a room temperature vacuum chamber. The dried samples were stored in a desiccator.

[0052] FIG. 1 schematically illustrates a cross-section of a drug delivery device manufactured using the coating solution of Example 1-1, wherein the drug delivery device comprises a microneedle; a drug layer coated on at least a portion of the surface of the microneedle; and a support supporting the microneedle, wherein the drug layer contains hyaluronidase and a hydrophilic polymer as a thickener.

[0053] Figure 2 is a photograph of a microneedle manufactured by coating twice with a coating solution using PVP (A), PVA (B), and HEC (C), respectively.

[0054] On the other hand, when manufacturing using HPMC or HPC, the coating solution gelled and it was impossible to coat the microneedles (see Fig. 4).

[0055] Experimental Example 1: Dissolution rate test using cadaver human skin

[0056] A dissolution rate test using cadaver skin was conducted on the drug delivery device manufactured in Example 1 as follows:

[0057] Cadaver human skin should be removed from the freezer 2 hours before use and thawed.

[0058] A hydrocolloid band was attached to the back of the microneedle patch and applied to cadaver skin with 50 N of pressure for approximately 10 seconds. The microneedles were then removed at intervals of 30 minutes, 1 hour, 1 hour 30 minutes, and 2 hours while maintaining a temperature similar to skin temperature of 32°C. The remaining surface was then observed under a stereoscopic microscope.

[0059] Figure 3 shows the results of a dissolution rate test of a drug delivery device according to the present invention observed under a microscope.

[0060] Observations showed that polyvinylpyrrolidone (PVP) and polyvinyl alcohol (PVA) dissolved relatively quickly within 30 minutes, while hydroxyethyl cellulose (HEC) dissolved relatively slowly (dissolved after about 1 hour).

[0061] Experimental Example 2: Measurement of shear viscosity using a rheometer for different types of thickeners (hydrophilic polymers)

[0062] In order to measure the shear viscosity of the coating solution of Example 1, a rotational rheometer (Discovery hybrid rheometer, HR-1, TA instrument, USA) was used to measure the shear viscosity, and the results are shown in Fig. 5.

[0063] To predict the rate of drug release from a solidified formulation in the body, viscosity was measured, and the results showed that HEC had the highest viscosity, followed by PVA and PVP. The bottom is the result of the measurement using water (blue) as a control group.

[0064] As with the results in Figure 3, the rate at which polymers dissolve in the body was the fastest for PVP and the slowest for HEC, and it was confirmed that the viscosity of the thickener affects the drug release rate.

[0065] Experimental Example 3: Dissolution Rate Test by Thickener Type

[0066] The dissolution test of the drug delivery device manufactured in Example 1 was conducted as follows:

[0067] A phosphate buffer solution (pH 7.4 phosphate buffer solution) was added to the receptor in a Franz-type diffusion cell, and the solution was stirred at a constant speed of 600 rpm using a magnetic stirrer while maintaining a temperature similar to skin temperature of 32°C to remove dissolved gases in the solution.

[0068] Afterwards, the drug delivery device of Example 1 was exposed to the phosphate buffer of the receptor so as to fit the upper donor cell of the Franz-type diffusion device, and then the solution of the receptor part was collected at predetermined intervals (after 5, 10, 15, and 20 minutes of being mounted on the Franz-type diffusion device), and a new buffer solution was replenished in the same amount as the collected amount. The amount of hyaluronidase present in the collected sample was analyzed using the content analysis method of "Hyaluronidase for injection" listed in the United States Pharmacopeia (USP).

[0069] <USP 26 for Hyaluronidase for Injection>

[0070] This method indirectly measures the activity of hyaluronidase by measuring the amount of hyaluronic acid (HA) that remains undecomposed after allowing hyaluronidase to react with HA at 37°C for 30 minutes, and analyzes it as turbidity.

[0071] 1) Add 500 ul of Hyaluronate solution to the test tube.

[0072] 2) To draw a standard concentration-response curve, add 500, 400, 300, 200, 100, and 0 ul of diluent, and then add 0, 100, 200, 300, 400, and 500 ul of standard solution at 30-second intervals. For the sample solution, dilute the sample collected in the above dissolution test 100-fold with the diluent, and then add 500 ul.

[0073] 3) Gently shake the solution containing the hyaluronate solution, standard, and sample solutions and leave it in a constant temperature water bath at 37 degrees for 30 minutes.

[0074] 4) Add 4 ml of serum solution at 30-second intervals, shake gently again, and leave at room temperature for 30 minutes.

[0075] 5) Shake well to mix before measurement and measure absorbance at 640 nm using a spectrophotometer.

[0076] The accumulated value of the amount of hyaluronidase present in the sample at each collection time point corresponds to the cumulative dissolution rate (%) of hyaluronidase released into the phosphate buffer among the hyaluronidase contained in the drug delivery device of the present invention.

[0077] Figure 6 is a graph depicting the cumulative dissolution rate of a drug delivery device according to the present invention. The cumulative dissolution rate and release rate were highest when PVP was used as a thickener (hydrophilic polymer). However, in terms of sustained efficacy, HEC or PVA appear to be more suitable.

[0078] However, PVA tends to aggregate during the coating process, making it difficult to apply it evenly to the microneedles.

[0079] Therefore, combining HEC and PVP can increase the initial release rate while also improving the duration of efficacy.

[0080] Figure 7 is a graph showing the cumulative dissolution rate when the hydrophilic polymers HEC and PVP were used alone and when they were used in combination.

[0081] When a mixture of HEC and PVP (blue-green) was used as a hydrophilic polymer, the initial drug release rate was lower than that of PVP, which had the highest cumulative release rate when mixed, but higher than that of HEC.

[0082] Therefore, the desired dissolution rate can be obtained by mixing HEC and PVP.

[0083] The drug delivery device according to the present invention has excellent transdermal drug delivery efficiency.

Claims

1. A composition for coating at least a portion of the surface of a microneedle in a microneedle formulation for transdermal administration of a drug; A composition comprising hyaluronidase and a hydrophilic polymer as active ingredients.

2. In the first paragraph, the composition is a hyaluronidase-containing composition effective for suppressing edema or treating inflammation.

3. A composition in claim 1, wherein hyaluronidase is contained in an amount of 20% or less relative to the total weight of the composition.

4. A composition according to claim 1, wherein the average molecular weight of the hydrophilic polymer material is 300 to 1,300 kDa.

5. A composition in paragraph 1, wherein the hydrophilic polymer material is at least one selected from the group consisting of HPMC, HPC, HEC, PVP, PVA (polyvinyl alcohol), and carboxymethyl cellulose.

6. A composition further comprising a surfactant according to claim 1.

7. A composition in claim 6, wherein the surfactant is at least one selected from the group consisting of poloxamer, sorbitan ester (span), and polysorbate (tween).

8. A drug delivery device for suppressing edema or treating inflammation, comprising a micro needle; and a support supporting the micro needle; A drug delivery device wherein at least a portion of the surface of the microneedle is coated with a drug layer comprising hyaluronidase as an active ingredient and a hydrophilic polymer.

9. A drug delivery device in claim 8, wherein the drug layer further comprises a surfactant.

10. In the 8th paragraph, the area of ​​the support is 1 to 25 cm 2 Human drug delivery device.

11. A drug delivery device according to claim 8, wherein the hydrophilic polymer material has an average molecular weight of 300 to 1,300 kDa.

12. A drug delivery device in claim 8, wherein the hydrophilic polymer material is at least one selected from the group consisting of HPMC, HPC, HEC, PVP, PVA (polyvinyl alcohol), and carboxymethyl cellulose.

13. A drug delivery device according to claim 8, characterized in that the hydrophilic polymer material is HEC, PVP or a mixture thereof.

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