Microstructure formulation techniques for botulinum toxin

Abstract

The present invention relates to microstructure formulation techniques for botulinum toxin. The microstructure formulation techniques for botulinum toxin according to the present invention make it possible to precisely control the concentration of botulinum toxin and to alleviate the pain occurring when botulinum toxin is administered into the human body, and also enable botulinum toxin to be accurately administered to a desired position. Thus, the present invention is expected to greatly contribute to the safe and convenient medical use of botulinum toxin.

Claims

1. A microstructure selected from the group consisting of a microneedle, a microblade, a microknife, a microfiber, a microspike, a microprobe, a microbarb, a microarray, and a microelectrode, wherein the microstructure is coated with a botulinum toxin composition comprising a botulinum toxin selected from the group consisting of botulinum toxin type A, B, C, D, E, F and G, a thickener and a stabilizer, wherein the thickener is any one or more selected from the group consisting of carboxymethyl cellulose sodium salt, sodium alginate, hyaluronic acid, methyl cellulose, hydroxyethyl cellulose, and polyvinyl pyrrolidone, and wherein the stabilizer is trehalose, sucrose, or a combination of trehalose and sucrose, wherein the microstructure is precoated with a polymer compound or polyvinyl alcohol.

2. A method for coating a microstructure with a botulinum toxin composition, the method comprising the steps of: (a) precoating the microstructure, wherein the microstructure is selected from the group consisting of a microneedle, a microblade, a microknife, a microfiber, a microspike, a microprobe, a microbarb, a microarray, and a microelectrode with a polymer compound or polyvinyl alcohol; (b) mixing a botulinum toxin selected from the group consisting of botulinum toxin type A, B, C, D, E, F and G, a thickener and a stabilizer to obtain a mixture; and (c) dipping the precoated microstructure of step (a) into the mixture of step (b), followed by drying, wherein the thickener is any one or more selected from the group consisting of carboxymethyl cellulose sodium salt, sodium alginate, hyaluronic acid, methyl cellulose, hydroxyethyl cellulose, and polyvinyl pyrrolidone, and wherein the stabilizer is trehalose, sucrose, or a combination of trehalose and sucrose.

Description

DESCRIPTION OF DRAWINGS

(1) The patent or application file contains at least one drawing executed in color. Copies of this patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

(2) FIG. 1 shows the results of measuring the percent recovery relative to the theoretical potency of botulinum toxin depending on the kind of precoating material in an example of the present invention.

(3) FIG. 2 shows the results of measuring the percent recovery relative to the theoretical potency of botulinum toxin depending on the kind and concentration of thickener mixed with botulinum toxin, in an example of the present invention.

(4) FIG. 3 shows the results of measuring the percent recovery relative to the theoretical potency of botulinum toxin depending on the kind and concentration of stabilizer mixed with the botulinum toxin, in an example of the present invention.

(5) FIG. 4 shows the results of measuring the long-term stabilities of combinations of a SA or HA thickener and a stabilizer in an example of the present invention.

(6) FIG. 5 shows the results of measuring the long-term stabilities of combinations of a PVP or HEC thickener and a stabilizer in an example of the present invention.

(7) FIG. 6 shows the results of confirming the coating uniformity of the coating composition containing a botulinum toxin.

BEST MODE

(8) The present invention is provided a coating composition, containing: a botulinum toxin; a thickener; and a stabilizer. In the coating composition, the botulinum toxin may be selected from the group consisting of botulinum toxin type A, B, C, D, E, F and G. The botulinum toxin is preferably botulinum toxin type A. The thickener may be any one or more selected from the group consisting of carboxymethylcellulose sodium salt, sodium alginate, hyaluronic acid, methyl cellulose, hydroxyethyl cellulose, and polyvinyl pyrrolidone, and the thickener may be contained in an amount of 0.05-10 wt %. The stabilizer may be any one or more selected from the group consisting of trehalose, methionine, sodium phosphate, and a mixture of human serum albumin and sodium chloride, and the stabilizer may be contained in an amount of 0.03-30 wt %. The coating composition may be coated on the surface of a microstructure, and the microstructure may be a microneedle, a microblade, a microknife, a microfiber, a microspike, a microprobe, a microbarb, a microarray or a microelectrode. The microstructure is preferably a microneedle.

Mode for Invention

(9) Hereinafter, the present invention will be described in further detail with reference to examples. It will be obvious to those skilled in the art that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

Example 1: Examination of Dilution Factor for Testing Botulinum Toxin Potency

(10) Because the loss rate of botulinum toxin potency, which occurs when a 2 coating composition and a 2 botulinum toxin composition are mixed with each other, cannot be seen, a preliminary experiment was performed in order to examine the change in botulinum toxin potency immediately after preparation of a botulinum toxin composition.

(11) For this, a 2 coating composition and a 2 botulinum toxin composition were prepared individually and mixed at a ratio of 1:1, thereby preparing mixture compositions as shown in Table 1 below.

(12) TABLE-US-00001 TABLE 1 Preparation Final concentration after mixing of 2 coating Example composition and 2 botulinum toxin composition HSA + NaCl 0.5% HSA, 0.9% NaCl, 5000 U toxin/6.5 l Tween 20 0.2% Tween 20, 5000 U toxin/6.5 l CMC 1% CMC, 5000 U toxin/6.5 l Sodium phosphate 0.025M sodium phosphate, 5000 U toxin/6.5 l (pH 6.0) HSA (human serum albumin) NaCl (sodium chloride) CMC (carboxymethylcellulose sodium salt) Sodium phosphate

(13) For the above-described mixture compositions, the potency of botulinum toxin was measured using an in vitro potency measurement kit (BoTest Matrix Botulinum Neurotoxin Detection Kits, Biosentinel, Inc.). Specifically, the measurement was performed in the same manner as described in Dunning F M et al., J Vis Exp. 2014 Mar. 3; (85). doi: 10.3791/51170. Furthermore, considering the case in which the potency would decrease immediately after preparation, two dilution factors (75 and 112.5) were applied. The results of the measurement are shown in Table 2 below.

(14) TABLE-US-00002 TABLE 2 Theoretical Potency Preparation potency Dilution Measured Average Recovery Example (U/6.25 l) factor Potency (U) potency SD RSD (%) HSA + NaCl 5000 75 6169.0 6240 100.7 1.6 124.8 112.5 6311.4 Tween 20 5000 75 6465.4 6561 135.2 2.1 131.2 112.5 6656.7 CMC 5000 75 3235.5 3427 270.9 7.9 68.5 112.5 3618.6 Sodium phosphate 5000 75 4275.5 4423 208.7 4.7 88.5 (pH 6.0) 112.5 4570.7

(15) The results of measurement of botulinum toxin potency for each of the mixture compositions indicated that the percent recovery relative to the theoretical potency was 50% or higher. However, it was shown that the percent recovery measured for CMC was out of the range of the standard curve when a dilution factor of 112.5 was applied, and that the percent recovery values for CMC and sodium phosphate immediately after preparation were lower than the value measured for HSA or Tween 20.

Example 2: Selection of Precoating Material

(16) To examine the change in the potency of the botulinum toxin according to whether a PLA (poly(lactic acid)) substrate is precoated and the kind of precoating material, an experiment was performed. 6.5 l of a coating composition comprising each of HSA+NaCl, Tween 20, CMC and sodium phosphate (pH 6.0), mixed with botulinum toxin, was dropped onto a PLA substrate (non-coated; N), a 2% PVA precoated substrate (PVA), or a corresponding thickener coated substrate (e.g., when 1% HSA+1.8% NaCl was used as the thickener, a PLA substrate was also coated with 1% HSA+1.8% NaCl), and then dried overnight at room temperature. After completion of the drying, each of the substrates was put in 0.3 mL of 0.9% NaCl contained in a 1.5 mL tube, and the toxin was eluted at room temperature for 30 minutes. After completion of the elution, considering the case in which the potency of the botulinum toxin would remain 100% during recovery after dropping, each composition was diluted with a dilution factor of 83.3 and subjected to in vitro potency measurement. The results of the measurement are shown in Table 3 below and FIG. 1.

(17) TABLE-US-00003 TABLE 3 Theoretical Measured Potency Preparation Substrate potency Dilution Potency recovery Example precoating (U) factor (U) (% ) HSA + N 5000 83.3 6195.2 123.9 NaCl PVA 5000 83.3 6811.5 136.2 Corresponding 5000 83.3 6020.7 120.4 thickener Tween 20 N 5000 83.3 N/D Low PVA 5000 83.3 N/D Low Corresponding 5000 83.3 N/D Low thickener CMC N 5000 83.3 N/D Low PVA 5000 83.3 5950.5 119.0 Corresponding 5000 83.3 N/D Low thickener Sodium N 5000 83.3 N/D Low phosphate PVA 5000 83.3 3883.3 77.7 (pH 6.0) (N/D; not detected)

(18) As shown in Table 3 above and FIG. 1, when the potency during recovery after dropping was higher than the upper limit of the standard curve, it was expressed as N/D. Considering the results for sodium phosphate, it was shown that, when the substrate was coated with 2% PVA, the potency was stably maintained.

(19) Tween 20 showed low potency in all the substrates, indicating that it is not suitable as a coating composition. In addition, CMC also showed no potency in all the substrates other than the PVA-precoated substrate, in the same manner as sodium phosphate.

Example 3: Selection of Optimum Thickener for Coating Composition Containing Botulinum Toxin

(20) To select a thickener to be contained in a coating composition from among candidate materials, each of eight coating candidate materials was prepared at 2 concentration (2% CMC, 1% SA, 1% HA, 1% MC, 1% HEC, 2% PVP, 1% HSA+1.8% NaCl, and 0.05M phosphate), and mixed with a botulinum toxin 2 concentration solution (5,000 U/3.25 l) at a ratio of 1:1. Then, 6.5 l of each of the prepared solutions was dropped onto a 2% PVA-precoated substrate, and then dried overnight at room temperature. After completion of the drying, each of the substrates was put in 0.3 mL of 0.9% NaCl contained in a 1.5 mL tube, and the toxin was eluted at room temperature for 30 minutes. After completion of the elution, considering the case in which the potency of the botulinum toxin would remain 100% during recovery after dropping, each coating solution was diluted with a dilution factor of 83.3 and subjected to in vitro potency measurement. The results of the measurement are shown in Table 4 below and FIG. 2.

(21) TABLE-US-00004 TABLE 4 Average of Average of Kind of Measured measured total coating potency potency Total Total recovery solution n (U) values SD RSD values (%) 1. CMC 1-1 4232.3 4004.4 177.7 4.4 80.1 1-2 4057.8 2-1 3877.9 2-2 3849.6 2. SA 1-1 3412.6 3356.6 51.4 1.5 67.7 1-2 3338.3 2-1 3381.0 2-2 3294.6 3. HA 1-1 3216.0 3193.5 72.5 2.3 63.9 1-2 3086.3 2-1 3245.7 2-2 3226.1 4. MC 1-1 4494.5 4507.2 10.1 0.2 90.1 1-2 4518.9 2-1 4509.8 2-2 4505.7 5. HEC 1-1 4627.1 4356.6 183.2 4.2 87.1 1-2 4286.5 2-1 4221.3 2-2 4291.4 6. PVP 1-1 4097.1 4137.5 83.1 2.0 82.8 1-2 4165.6 2-1 4048.2 2-2 4239.2 7. Sodium 1-1 2915.0 2891.0 81.4 2.8 57.8 phosphate 1-2 2787.1 2-1 2982.6 2-2 2879.5 8. HSA + 1-1 5044.6 4769.7 216.7 4.5 95.4 NaCl 1-2 4837.7 2-1 4567.2 2-2 4629.2 CMC (carboxymethylcellulose sodium salt) SA (sodium alginate) HA (hyaluronic acid 1,000 kDa) MC (methyl cellulose) HEC (hydroxyethyl cellulose) PVP (polyvinyl pyrrolidone) Sodium phosphate

(22) Two samples (sample 1 and 2) per test group were prepared, and toxin eluted from each of the samples was measured twice. As a result, as can be seen in Table 4 above, it was shown that, among the coating compositions, the percent recovery relative to the theoretical potency of each of CMC, MC, HEC, PVP, and HSA+NaCl was as high as 80% or above. Particularly, MC and HSA+NaCl showed a high potency recovery of 90% or above. In addition, SA and HA also showed an average potency recovery of 60% or above, which was higher than that shown by sodium phosphate.

Example 4: Selection of Optimum Stabilizer for Coating Composition Containing Botulinum Toxin

(23) To select a stabilizer to be contained in a coating composition among candidate materials, each of coating compositions prepared as shown in Table 5 below was mixed with a botulinum toxin 2 concentration solution (5,000 U/3.25 l) at a ratio of 1:1, and 6.5 l of each of the prepared solutions was dropped onto a 2% PVA-precoated substrate, and then dried at room temperature overnight. After completion of drying, each of the substrates was placed in 0.3 mL of 0.9% NaCl contained in a 1.5 mL tube, and the toxin was eluted at room temperature for 30 minutes. After completion of elution, considering the case in which the potency of the botulinum toxin remains 100% during recovery after dropping, each coating solution was diluted at a factor of 83.3 and subjected to in vitro potency measurement.

(24) TABLE-US-00005 TABLE 5 Preparation Example 2 concentration 1. PVP + Tre 10% 2% PVP, 20% Tre 2. PVP + Tre 15% 2% PVP, 30% Tre 3. PVP + Tre 20% 2% PVP, 40% Tre 4. PVP + Met 10 Mm 2% PVP, 20 mM Met 5. PVP + Met 20 mM 2% PVP, 40 mM Met 6. PVP + Met 30 mM 2% PVP, 60 mM Met 7. HEC + Tre 10% 1% HEC, 20% Tre 8. HEC + Tre 15% 1% HEC, 30% Tre 9. HEC + Tre 20% 1% HEC, 40% Tre 10. HEC + Met 10 mM 1% HEC, 20 mM Met 11. HEC + Met 20 mM 1% HEC, 40 mM Met 12. HEC + Met 30 mM 1% HEC, 60 mM Met 13. CMC + Tre 10% 2% CMC, 20% Tre 14. CMC + Tre 15% 2% CMC, 30% Tre 15. CMC + Tre 20% 2% CMC, 40% Tre 16. CMC + Met 10 mM 2% CMC, 20 mM Met 17. CMC + Met 20 mM 2% CMC, 40 mM Met 18. CMC + Met 30 mM 2% CMC, 60 mM Met 19. HSA + NaCl 1% HSA, 1.8% NaCl 20. Sodium phosphate 0.05M sodium phosphate Tre (trehalose) Met (methionine)

(25) The experimental results in Table 6 indicated that PVP and HEC other than CMC showed a potency recovery of 90% or more relative to the value measured immediately after HSA preparation, in all stabilizer conditions. The results are shown in FIG. 3.

Example 5: Examination of Accelerated Stability of Coating Composition Containing Botulinum Toxin

(26) The long-term stabilities of mixtures of thickeners (SA, HA, PVP and HEC) and stabilizers (Tre and Met), selected based on the results of Examples 1 to 4 above, were examined.

(27) To test the long-term stabilities of combinations of a SA or HA thickener and a stabilizer, PLA substrates were precoated with 2% PVA, and each of coating compositions prepared as shown in Table 6 below was mixed with a botulinum toxin 2 concentration solution (5,000 U/3.25 l) at a ratio of 1:1. 6.5 l of each of the mixtures was dropped onto each of the PLA substrates, and then dried overnight at room temperature. After completion of the drying, each of the substrates was put in 0.3 mL of 0.9% NaCl contained in a 1.5 mL tube, and the percent potency recovery for each substrate was measured up to day 8. Because the coating compositions themselves, which comprise mixtures of thickeners and stabilizers in various combinations, would affect the measurement of the botulinum toxin potency, the potency measured for each test group on day 1 was taken as a reference value 100, and the potency measured on day 8 was expressed relative to the potency measured on day 1. The results are shown in FIG. 4. The test results indicated that most of the test groups comprising the SA or HA thickener maintained a potency of 50% or higher relative to the day-1 potency up to day 8.

(28) TABLE-US-00006 TABLE 6 Preparation Example Remarks 1. SA + Tre 10% 0.5% Sodium Alginate, 10% Trehalose 2. SA + Tre 15% 0.5% Sodium Alginate, 15% Trehalose 3. HA + Tre 10% 0.5% Hyaluronic acid, 10% Trehalose 4. HA + Tre 15% 0.5% Hyaluronic acid, 15% Trehalose 5. HA + Tre 20% 0.5% Hyaluronic acid, 20% Trehalose 6. HA + Met 10 mM 0.5% Hyaluronic acid, 10 mM Methionine 7. HA + Met 20 mM 0.5% Hyaluronic acid, 20 mM Methionine 8. HA + Met 30 mM 0.5% Hyaluronic acid, 30 mM Methionine 9. PVP + Tre 10% 1% Polyvinyl pyrrolidone, 10% Trehalose 10. PVP + Met 20 mM 1% Polyvinyl pyrrolidone, 20 mM Methionine 11. HAS + NaCl 0.5% Human Serum Albumin, 0.9% Sodium Chloride 12. NaP 25 mM Sodium Phosphate pH 6.0

(29) In addition, in order to test the long-term stabilities of combinations of a PVP or a HEC thickener and a disaccharide or an amino acid stabilizer, botulinum toxin coating compositions were prepared and dropped in the same manner as the test performed on the SA and HA thickeners, and then an accelerated stability test was performed at 37 C. for 5 weeks. Trehalose (Tre), or sucrose (Suc) was used as a disaccharide stabilizer, and methionine (Met) or glycine (Gly) was used as an amino acid stabilizer. For an accurate comparison of measured values, the potency immediately after preparation of each of the samples was taken as a reference value (100%), and the potencies at varying time points during 5 weeks were expressed relative to the reference value. In addition, the value of a standard product that is a positive control in the BoTest test was applied to minimize the error between the tests. The results of the test are shown in Table 7 below and FIG. 5.

(30) TABLE-US-00007 TABLE 7 Immediately after 1 8 22 36 Preparation Example preparation day days days days 1% PVP 100.0 92.3 71.2 61.1 N/D 1% PVP + 10% Tre 100.0 107.9 85.1 70.2 57.2 1% PVP + 30% Suc 100.0 239.5 154.7 154.7 68.1 1% PVP + Met 20 mM 100.0 108.4 105.9 87.7 55.0 1% PVP + Gly 20 mM 100.0 79.1 N/D N/D N/D 0.5% HEC 100.0 80.7 65.2 N/D N/D 0.5% HEC + 10% Tre 100.0 92.5 88.9 66.0 53.4 0.5% HEC + 30% Suc 100.0 81.6 55.7 58.9 53.1 0.5% HEC + Met 20 mM 100.0 104.0 59.9 N/D N/D 0.5% HEC + Gly 20 mM 100.0 83.2 N/D N/D N/D HSA +NaCl 100.0 112.7 86.0 61.5 45.6 Sodium phosphate (pH 6.0) 100.0 105.2 89.4 67.9 N/T (N/D: not detected, N/T: not tested)

(31) The test results indicated that the combination using PVP, or HEC as a thickener, and the disaccharide, or methionine as the stabilizer are the optimal stabilized combinations for preparation of botulinum toxin coating compositions, compared to the positive control HSA.

Example 6: Confirmation of Coating Uniformity of Coating Composition Containing Botulinum Toxin

(32) Coating uniformity of compositions for coating on substrates such as microneedles is very important. In addition, since the composition comprises botulinum toxin, the titer stability of the botulinum toxin should be ensured. Thus, a substrate was precoated with 2% PVA, the substrate was coated with a thickener and stabilizer combination composition which was evaluated as having excellent titer stability of botulinum toxin in Example 5, and coating uniformity was evaluated by average number of pixels and magenta ratio. More specifically, the picture was set to CMYK instead of RGB, the size of the needle was equally adjusted in each picture, the back background of the picture was deleted except needle, the total area of the needle was measured, and the average number of pixels and magenta ratio were measured in the stained area of the needle. The results are shown in Table 8, Table 9, and FIG. 6.

(33) TABLE-US-00008 TABLE 8 Preparation Example Average number of pixels HEC + Suc, once for 10 seconds 392 PVP + Tre, once for 10 seconds 366 HEC + Suc, once for 15 seconds 605 PVP + Tre, once for 15 seconds 402

(34) TABLE-US-00009 TABLE 9 Preparation Example Magenta ratio (%) HEC + Suc, once for 10 seconds 84 90 88 92 89 93 PVP + Tre, once for 10 seconds 47 81 66 81 71 79

(35) The experimental results showed that the HEC+Suc combination had a higher average number of pixels than the PVP+Tre combination when the microneedle was coated for 10 or 15 seconds. In addition, the ratio of magenta dyed area to total microneedle area was significantly higher in the HEC+Suc combination when the same time was applied (The average of HEC+Suc combination: 89%, and the average of PVP+Tre combination: 71%). As a result, it was found that the coating using the HEC+Suc combination is more uniform than using the PVP+Tre combination.

(36) From the results of Examples 1 to 6 above, it could be found that, in fabrication of stabilized microstructures enabling a botulinum toxin to be accurately administered to a desired position at a precisely controlled concentration without causing discomforts such as pain. Carboxymethyl cellulose sodium salt, sodium alginate, hyaluronic acid, methyl cellulose, hydroxyethyl cellulose, polyvinyl pyrrolidone, or combinations thereof are effective as thickeners. Furthermore, trehalose, sucrose, a combination of trehalose and sucrose, methionine, sodium phosphate, a mixture of human serum albumin and sodium chloride, or combinations thereof are remarkably effective as stabilizers.

INDUSTRIAL APPLICABILITY

(37) The present invention relates to microstructure formulation techniques for botulinum toxin. The microstructure formulation techniques for botulinum toxin according to the present invention make it possible to precisely control the concentration of botulinum toxin and to alleviate the pain occurring when botulinum toxin is administered into the human body, and also enable botulinum toxin to be accurately administered to a desired position. Thus, the present invention is expected to greatly contribute to the safe and convenient medical use of botulinum toxin.