METHOD FOR INCREASING CELL VIABILITY BY USING DRUG DELIVERY SYSTEM AND ULTRASOUND IRRADIATION, AND ULTRASOUND IRRADIATION APPARATUS USING SAME

Abstract

Disclosed are a method for increasing the viability of cells by ultrasound irradiation and an ultrasound irradiation apparatus using same. The method comprises: applying a drug and an epidermal drug delivery system onto the epidermis of a subject; and, with ultrasound parameters each being preset within a certain range, an ultrasound irradiation apparatus irradiating ultrasound onto the epidermis of the subject through an ultrasound generation unit placed within a critical range from the epidermis of the subject, wherein the epidermal drug delivery system induces the formation of cavities, i.e., cavitation around the epidermis of the subject when the ultrasound is irradiated, the ultrasound parameters at least include an ultrasound pressure and an ultrasound duty percentage, the ultrasound pressure is 0.5-1 MPa, and the ultrasound duty percentage is 1-5%.

Claims

1. A method for increasing a viability of one or more cells by irradiating the cells with ultrasound, comprising a step of: on condition that a drug and an epidermal drug carrier are applied on epidermis of a subject and that ultrasound parameters have been preset within respective ranges, an ultrasound irradiating device positioning an ultrasonic transducer within a threshold range from the epidermis of the subject and then irradiating the epidermis with the ultrasound, wherein the epidermal drug carrier causes cavitation to create at least one cavity around the epidermis in response to irradiating the epidermis with the ultrasound, and wherein the ultrasound parameters include pressure of the ultrasound and duty percentage of the ultrasound, wherein the pressure of the ultrasound ranges from 0.5 MPa to 1 MPa, and wherein the duty percentage of the ultrasound ranges from 1% to 5%.

2. The method of claim 1, wherein the ultrasound parameters further include a pressure of the ultrasound, a duty percentage of the ultrasound, and an intensity of the ultrasound, and wherein the pressure ranges from 0.5 MPa to 1 MPa, the duty percentage ranges from 1% to 5%, and the intensity ranges from 166.7 mW/cm.sup.2 to 416.7 mW/cm.sup.2.

3. The method of claim 2, wherein the drug is Redensyl.

4. The method of claim 3, wherein a concentration of the Redensyl ranges from 0.5% to 1%.

5. The method of claim 1, wherein the ultrasound parameters further include frequency of the ultrasound, and wherein the frequency of the ultrasound ranges from 0.5 MHz to 4.6 MHz.

6. The method of claim 5, wherein the frequency of the ultrasound is 1 MHz.

7. The method of claim 1, wherein the ultrasound parameters further include total irradiation time of the ultrasound, and wherein the total irradiation time is equal to or less than ten minutes.

8. The method of claim 1, wherein the cells are outer root sheath cells.

9. An ultrasound irradiating device for increasing a viability of one or more cells by irradiating the cells with ultrasound, comprising: an ultrasound transducer; and a controlling part, on condition that a drug and an epidermal drug carrier are applied on epidermis of a subject and that ultrasound parameters have been preset within respective ranges, for positioning the ultrasonic transducer within a threshold range from the epidermis of the subject and then allowing the ultrasonic transducer to irradiate the epidermis with the ultrasound; and wherein the epidermal drug carrier causes cavitation to create at least one cavity around the epidermis in response to irradiating the epidermis with the ultrasound, and wherein the ultrasound parameters include a pressure of the ultrasound and a duty percentage of the ultrasound, wherein the pressure of the ultrasound ranges from 0.5 MPa to 1 MPa, and wherein the duty percentage of the ultrasound ranges from 1% to 5%.

10. The ultrasound irradiating device of claim 9, wherein the ultrasound parameters further include a pressure of the ultrasound, a duty percentage of the ultrasound, and an intensity of the ultrasound, and wherein the pressure ranges from 0.5 MPa to 1 MPa, the duty percentage ranges from 1% to 5%, and the intensity ranges from 166.7 mW/cm.sup.2 to 416.7 mW/cm.sup.2.

11. The ultrasound irradiating device of claim 10, wherein the drug is Redensyl.

12. The ultrasound irradiating device of claim 11, wherein a concentration of the Redensyl ranges from 0.5% to 1%.

13. The ultrasound irradiating device of claim 9, wherein the ultrasound parameters further include frequency of the ultrasound, and wherein the frequency of the ultrasound ranges from 0.5 MHz to 4.6 MHz.

14. The ultrasound irradiating device of claim 13, wherein the frequency of the ultrasound is 1 MHz.

15. The ultrasound irradiating device of claim 9, wherein the ultrasound parameters further include total irradiation time of the ultrasound, and wherein the total irradiation time is equal to or less than ten minutes.

16. The ultrasound irradiating device of claim 9, wherein the cells are outer root sheath cells.

Description

4. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] FIGS. 1A and 1B are drawings schematically illustrating ultrasound parameters used in a method for increasing viability of one or more cells by irradiating the cells with ultrasound in accordance with one example embodiment of the present disclosure.

[0032] FIG. 2 is a drawing schematically illustrating experimental results of a dermal stability of the method for increasing the viability of the cells by irradiating the cells with the ultrasound in accordance with one example embodiment of the present disclosure.

[0033] FIGS. 3A and 3B are drawings schematically illustrating the viability of human outer root sheath cells according to concentrations of applied conventional drugs in case only the conventional drugs are applied.

[0034] FIG. 4 is a drawing schematically illustrating an epidermal drug carrier used in the method for increasing the viability of the cells by irradiating the cells with the ultrasound in accordance with one example embodiment of the present disclosure.

[0035] FIGS. 5 to 21 are drawings schematically illustrating physical effects of the ultrasound while varying the ultrasound parameters in accordance with one example embodiment of the present disclosure.

[0036] FIGS. 22 to 25 are drawings schematically illustrating comparisons between experimental results of the viability of the human outer root sheath cells in accordance with one example embodiment of the present disclosure and experimental results of the viability of the human outer root sheath cells where only a drug is applied.

[0037] FIGS. 26 to 37 are drawings schematically illustrating experimental results of gene expression in response to irradiating the cells with the ultrasound in accordance with one example embodiment of the present disclosure.

[0038] FIGS. 38 to 41 are drawings schematically illustrating degrees of DNA methylation in response to irradiating the cells with the ultrasound in accordance with one example embodiment of the present disclosure.

[0039] FIG. 42 is a drawing schematically illustrating clinical results according to a lapse of time when the epidermal drug carrier, the drug, and the ultrasound are applied together for 10 minutes two times a week in accordance with one example embodiment of the present disclosure.

[0040] FIG. 43A is a drawing schematically illustrating a case where no treatment is applied.

[0041] FIG. 43B is a drawing schematically illustrating a case where a conventional treatment is applied for 12 months.

[0042] FIGS. 43C and 43D are drawings schematically illustrating clinical results according to a lapse of time when the epidermal drug carrier, the drug, and the ultrasound are applied together in accordance with one example embodiment of the present disclosure.

[0043] FIGS. 44A and 44B are drawings schematically illustrating clinical results according to a lapse of time before and after the epidermal drug carrier, the drug, and the ultrasound are applied together in accordance with one example embodiment of the present disclosure.

5. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0044] In the following detailed description, reference is made to the accompanying drawings that show, by way of illustration, specific embodiments in which the present disclosure may be practiced. These embodiments are described in sufficient detail to enable those skilled in the art to practice the present disclosure. It is to be understood that the various embodiments of the present disclosure, although different, are not necessarily mutually exclusive. For example, a particular feature, structure, or characteristic described herein in connection with one embodiment may be implemented within other embodiments without departing from the spirit and scope of the present disclosure. In addition, it is to be understood that the position or arrangement of individual elements within each disclosed embodiment may be modified without departing from the spirit and scope of the present disclosure. The following detailed description is, therefore, not to be taken in a limiting sense, and the scope of the present disclosure is defined only by the appended claims, appropriately interpreted, along with the full range of equivalents to which the claims are entitled. In the drawings, like numerals refer to the same or similar functionality throughout the several views.

[0045] To allow those skilled in the art to carry out the present disclosure easily, the example embodiments of the present disclosure will be explained in detail by referring to attached diagrams as shown below.

[0046] FIGS. 1A and 1B are drawings schematically illustrating ultrasound parameters used in a method for increasing viability of cells by irradiating the cells with ultrasound in accordance with one example embodiment of the present disclosure.

[0047] The ultrasound parameters are as follows.

Frequency: a frequency of the ultrasound
Duty percentage: a percentage value of actual time of ultrasound irradiation divided by a period
PRF (pulse repetition frequency): the number of times square wave is irradiated per second
PRP (pulse repetition period): 1/PRF
Pressure: a pressure of the ultrasound
Intensity=(duty percentage)×pressure.sup.2/(2×c×rho): energy of the ultrasound irradiated per unit area (c: speed of sound in water=1,500 m/s, rho: density of water=1,000 kg/m.sup.3)

[0048] As an example, in FIG. 1A, the square wave of the ultrasound with the frequency of 1 MHz is repeated three times per second, thus PRF is 3 Hz and PRP, i.e., 1/PRF, is 0.3333 second. Herein, a cycle may be a time between an instantaneous peak and its neighboring instantaneous peak in an envelope. Also, in FIG. 1B, the duty percentage is Ton/(Ton+Toff)×100, i.e., a time Ton, during which the actual ultrasound is irradiated, divided by the period Ton+Toff of the ultrasound irradiation.

[0049] For reference, a Table 1 below describes intensities according to pressures and duty percentages of the ultrasound.

TABLE-US-00001 TABLE 1 Intensity with Intensity with Intensity with Intensity with Intensity with 100% duty 1% duty 2% duty 3% duty 5% duty Pressure Intensity percentage percentage percentage percentage percentage (MPa) (W/m.sup.2) (W/cm.sup.2) (mW/cm.sup.2) (mW/cm.sup.2) (mW/cm.sup.2) (mW/cm.sup.2) 0.5 83333.3 8.3 83.3 166.7 250.0 416.7 0.7 163333.3 16.3 163.3 326.7 490.0 816.7 1 333333.3 33.3 333.3 666.7 1000.0 1666.7

[0050] Next, FIG. 2 is a drawing schematically illustrating experimental results of a dermal stability of the method for increasing the viability of the cells by irradiating the cells with the ultrasound in accordance with one example embodiment of the present disclosure.

[0051] Specifically, as a result of monitoring temperature changes during the ultrasound irradiation on a skin of an 8-week-old male rat, it is observed that a skin temperature increased by about 1.2 degrees Celsius after 30 minutes and that 0.05 degrees Celsius per minute was changed in temperature on average. That is, it can be seen that the degree of temperature change due to the ultrasound irradiation does not reach as much extent as it damages the skin.

[0052] Next, experimental conditions for observing the viability of the cells according to the ultrasound irradiation are described.

[0053] For reference, a Table 2 below describes cell seeding densities.

TABLE-US-00002 TABLE 2 Recommended 5*10.sup.3 cells/cm.sup.2 seeding density Surface Dish area (cm.sup.2) Seeding density  60 mm 21 1.05*10.sup.5 cells 100 mm 55 2.75*10.sup.5 cells T75 75 3.75*10.sup.5 cells

[0054] Also, a Table 3 below describes required amounts of the cells according to the conditions.

TABLE-US-00003 TABLE 3 Number of Ultrasound is irradiated per Number of cells seeding T75 well for 10 minutes wells (total amount) flask Cell 96 well plate Condition 22  6.6*10.sup.4 8.4*10.sup.6 viability (3*10.sup.3 cell/ 3 wells 66 1.98*10.sup.5 assay well) 6 well plate Condition 20 1.6 × 10.sup.6 (8*10.sup.4 cell/ 3 wells 60 4.8 × 10.sup.6 well) PCR 6 well plate Condition 22 1.76 × 10.sup.6  for drug Condition for 20 1.6 × 10.sup.6 ultrasound 3 wells 126 .sup. 1 × 10.sup.7

[0055] First, to explain the material, a cell culture is comprised of human hair outer root sheath cells (HHORSC), a mesenchymal stem cell medium (MSCM), an FBS 0.25% trypsin/EDTA solution, a trypsin neutralization solution, a Dulbecco's phosphate-buffered saline (DPBS), and poly-L-lysine.

[0056] And a WST-1 cell proliferation assay system is used for WST-1 assay. Further, Trizol, a sensiFAST probe Hi-ROX one step kit, PrimeTime qPCR assay are used for PCR.

[0057] First, for culture dish coating (based on a T75 flask), 10 mL of deionized water (D.W.) is put into the T75 flask and then 15 μL of poly-L-lysine (10 mg/mL) is put into the T75 flask. Then, the T75 flask is placed in an incubator at 37° C. and an inside of the T75 flask is coated with poly-L-lysine for 1 hour. And the T75 flask is washed twice with D.W.

[0058] And a whole medium is created by using MSCM consisting of 500 mL basal medium, 25 mL FBS, and 5 mL of mesenchymal stem cells.

[0059] And, a frozen cell vial is warmed in a water bath at 37° C. Then the melted cells are placed on a growth medium of 3 mL and centrifuged at 3,000 rpm for 3 minutes to pull the cells down to the bottom. Then, DPBS is added to the centrifuged cells, and after washing, centrifuged again at 3,000 rpm for 3 minutes to pull the cells down, and these processes are repeated twice, creating cell pellets. Then, 8 mL of the growth medium is put into the T75 flask coated with poly-L-lysine, the cell pellets are dissolved therein, and after seeding, the T75 flask is placed in an incubator at 37° C. with 5% CO.sub.2.

[0060] And, if it is confirmed under the microscope that the cells have grown more than 90% in the cell subculture of the T75 flask, (i) 3 mL of DPBS is added to the cell subculture and washed and (ii) 3 mL of the trypsin/EDTA solution is added and the T75 flask is placed in the incubator at 37° C. for 3 minutes. Then, if it is confirmed that the cells have fallen from the T75 flask, (i) each 3 mL of the growth medium and the trypsin neutralization solution is added to the T75 flask in order to neutralize the trypsin/EDTA solution, (ii) the neutralized cell solution is transferred to a conical tube, and centrifuged at 3,000 rpm for 3 minutes to pull the cells down, creating cell pellets, (iii) 8 mL of the growth medium is put into the T75 flask coated with poly-L-lysine, (iv) the cell pellets are dissolved in the T75 flask, and (v) after seeding, the T75 flask is placed in the incubator at 37° C. with 5% CO.sub.2.

[0061] And, for 6 well plate/96 well plate cell seeding, poly-L-lysine is added for each plate according to a Table 4 below, and poly-L-lysine coating is performed according to a culture dish coating procedure.

TABLE-US-00004 TABLE 4 Required amount of poly-L- lysine per surface area (μg/cm.sup.2) Required amount 2 Surface area of poly-L-lysine Poly-L-lysine Dish (cm.sup.2) (μg) (μL) of 10 mg/mL T75 75 150 15 100 mm 55 110 11 6 wells 4.8 9.6 2 12 wells 3.9 7.8 0.78 (7.8 μL as a result of 1/10 dilution) 96 wells 0.3 0.6 0.06 (6 μL as a result of 1/100 dilution)

[0062] Herein, if it is confirmed under the microscope that the cells have grown more than 90% in the cell subculture of the T75 flask, (i) 3 mL of DPBS is added to the cell subculture and washed and (ii) 3 mL of the trypsin/EDTA solution is added and the T75 flask is placed in the incubator at 37° C. for 3 minutes. Then, if it is confirmed that the cells have fallen from the T75 flask, (i) each 3 mL of the growth medium and the trypsin neutralization solution is added to the T75 flask in order to neutralize the trypsin/EDTA solution, (ii) the neutralized cell solution is transferred to the conical tube, and centrifuged at 3,000 rpm for 3 minutes to pull the cells down, and (iii) the cells are counted using trypan blue and a hemocytometer. And the cells are seeded as many as 8×10.sup.4 in the 6 well plate and 4×10.sup.4 in the 96 well plate.

[0063] For WST assay, in a case of an experiment of the ultrasound irradiation only, the cells seeded in the 6 well plate are cultured for 12 to 18 hours and observed with the microscope. And after removing the medium, the cells are washed twice with DPBS, put into a growth factor free medium, and irradiated with the ultrasound for 10 minutes. Then, the 6 well plate is placed in the incubator at 37° C. with 5% CO.sub.2 and incubated for 24 hours, then the medium is removed therefrom. The 6 well plate is then washed with DPBS, treated with 0.2 mL of WST-1, and placed in the incubator for 3 hours. Then supernatant is transferred from each well of the 6 well plate into 3 wells of a 96 well plate, and the 96 well plate is measured with a microplate reader at 450 nm.

[0064] For the WST assay, in a case of an experiment with drugs only, the cells seeded in the 96 well plate are cultured for 12 to 18 hours and confirmed whether the cells are seeded sufficiently. And after removing the medium, the cells are washed twice with DPBS, put into the growth factor free medium, and a drug is applied to the cells. Then, the 96 well plate is placed in the incubator at 37° C. with 5% CO.sub.2 for 24 hours, then the medium is removed therefrom. The 96 well plate is then washed with DPBS, treated with 0.2 mL of WST-1, and placed in the incubator for 3 hours. Then the 96 well plate is measured with the microplate reader at 450 nm.

[0065] Meanwhile, FIGS. 3A and 3B are drawings schematically illustrating the viability of the human outer root sheath cells according to concentrations of applied conventional drugs in case only the conventional drugs are applied.

[0066] Specifically, FIG. 3A shows the viability of the cells according to the concentrations of applied Redensyl, i.e., a hair loss treatment drug, and FIG. 3B shows the viability of the cells according to the concentrations of applied minoxidil, i.e., another hair loss treatment drug.

[0067] For reference, a Table 5 below shows experimental conditions where the drug, the epidermal drug carrier, and the ultrasound are applied together. Herein, a solubility of minoxidil is 0.2% in PBS, on a condition of being measured 24 hours after application of the drug. For reference, blanks in the Table 5 represent that a control group, where only the ultrasound is used, is unavailable due to cell deformation caused by too strong an ultrasound.

TABLE-US-00005 TABLE 5 96 well plate 6 well plate Positive Ultrasound + epidermal control drug carrier + Redensyl Control (EGF) Minoxidil Redensyl Ultrasound Ultrasound Microbubble Redensyl Medium 50 ng/ 0.00002% 0.01%   0.3 MPa 0.5 MPa 2% 1:1000 0.04%  100 μL 1% 0.1% 0.2% 0.0002% 0.02%   0.5% .sup. 1% 0.002% 0.04%   .sup. 2% .sup. 3% 0.005% 0.1%.sup.  0.5 MPa 0.5 MPa 5% 0.04%  2% 0.1% 0.2% 0.01% 0.2%.sup.  0.5% .sup. 1% 0.02% 0.5%.sup.  .sup. 2% .sup. 3% 0.05% 1% 1 MPa 1 MPa 1% 0.04%  2% 0.1% 0.2% 0.15% 2% 0.5% .sup. 1% 0.2% 3% .sup. 2% .sup. 3% 4% 1 MPa 3% 0.04%  0.1% 0.2% 5% 0.5% .sup. 1% 10%  .sup. 2% .sup. 3%

[0068] Next, FIG. 4 is a drawing schematically illustrating the epidermal drug carrier in accordance with one example embodiment of the present disclosure.

[0069] As an example, the epidermal drug carrier may be configured with a single membrane as shown in FIG. 4, but the scope of the present disclosure is not limited thereto, and may be formed of a plurality of membranes such as an outer membrane and an inner membrane, etc., or may have a structure incorporating a drug inside the membrane.

[0070] Meanwhile, in the cell experiment, a comparison between MTT and WST-1 was performed, and in the case of MTT, there was a problem of overlapping between an absorbance range of Redensyl and an absorbance range of MTT, resulting in a poor data accuracy. On the other hand, in the case of WST-1, since the absorbance range of Redensyl and the absorbance range of WST-1 did not overlap each other, the experiment was conducted by using WST-1.

[0071] In addition, a media comparison was performed. In a first experiment, after seeding stabilization, complete media was treated with Redensyl and observed for 24 hours. However, a time duration is not limited to 24 hours, and may be changed according to cell conditions. Also, in a second experiment, after the seeding stabilization, the media without a growth factor were treated with Redensyl and observed for 24 hours. Like the first experiment, the time duration of the second experiment may be changed according to the cell conditions.

[0072] By referring to the first experiment and the second experiment, an effect of the ultrasound was better observed when the media did not contain the growth factor. Therefore, the experiment was conducted by treating the media, which did not contain the growth factor, with Redensyl.

[0073] Subsequently, the ultrasound irradiation was conducted, and a quantitative analysis was performed through an assay before and after the ultrasound irradiation.

[0074] Tables 6 to 9 describe the experimental conditions of the ultrasound irradiation for evaluating physical effects of the ultrasound on the cells.

[0075] Specifically, Tables 6 and 7 describe observation conditions of Redensyl treatment on serum free media and complete media left overnight during the stabilization after the seeding.

TABLE-US-00006 TABLE 6 96Well-1 A B C D E F G H 1 M-24- M-24- M-24- M-24- M-24- M-24- M-24- M-24- XTT XTT XTT XTT XTT XTT XTT XTT 2 P-24-XTT P-24-XTT P-24-XTT P-24-XTT P-24-XTT P-24-XTT P-24-XTT P-24-XTT 3 R0.01- R0.01- R0.01- R0.01- R0.01- R0.01- R0.01- R0.01- 24-XTT 24-XTT 24-XTT 24-XTT 24-XTT 24-XTT 24-XTT 24-XTT 4 R0.04- R0.04- R0.04- R0.04- R0.04- R0.04- R0.04- R0.04- 24-XTT 24-XTT 24-XTT 24-XTT 24-XTT 24-XTT 24-XTT 24-XTT 5 R0.2- R0.2- R0.2- R0.2- R0.2- R0.2- R0.2- R0.2- 24-XTT 24-XTT 24-XTT 24-XTT 24-XTT 24-XTT 24-XTT 24-XTT 6 R1-24- R1-24- R1-24- R1-24- R1-24- R1-24- R1-24- R1-24- XTT XTT XTT XTT XTT XTT XTT XTT 7 M-24- M-24- M-24- M-24- M-24- M-24- M-24- M-24- WST WST WST WST WST WST WST WST 8 P-24- P-24- P-24- P-24- P-24- P-24- P-24- P-24- WST WST WST WST WST WST WST WST 9 R0.01- R0.01- R0.01- R0.01- R0.01- R0.01- R0.01- R0.01- 24-WST 24-WST 24-WST 24-WST 24-WST 24-WST 24-WST 24-WST 10 R0.04- R0.04- R0.04- R0.04- R0.04- R0.04- R0.04- R0.04- 24-WST 24-WST 24-WST 24-WST 24-WST 24-WST 24-WST 24-WST 11 R0.2- R0.2- R0.2- R0.2- R0.2- R0.2- R0.2- R0.2- 24-WST 24-WST 24-WST 24-WST 24-WST 24-WST 24-WST 24-WST 12 R1-24- R1-24- R1-24- R1-24- R1-24- R1-24- R1-24- R1-24- WST WST WST WST WST WST WST WST

TABLE-US-00007 TABLE 7 96Well-2 A B C D E F G H 1 M-48- M-48- M-48- M-48- M-48- M-48- M-48- M-48- XTT XTT XTT XTT XTT XTT XTT XTT 2 P-48- P-48- P-48- P-48- P-48- P-48- P-48- P-48- XTT XTT XTT XTT XTT XTT XTT XTT 3 R0.01- R0.01- R0.01- R0.01- R0.01- R0.01- R0.01- R0.01- 48-XTT 48-XTT 48-XTT 48-XTT 48-XTT 48-XTT 48-XTT 48-XTT 4 R0.04- R0.04- R0.04- R0.04- R0.04- R0.04- R0.04- R0.04- 48-XTT 48-XTT 48-XTT 48-XTT 48-XTT 48-XTT 48-XTT 48-XTT 5 R0.2- R0.2- R0.2- R0.2- R0.2- R0.2- R0.2- R0.2- 48-XTT 48-XTT 48-XTT 48-XTT 48-XTT 48-XTT 48-XTT 48-XTT 6 R1-48- R1-48- R1-48- R1-48- R1-48- R1-48- R1-48- R1-48- XTT XTT XTT XTT XTT XTT XTT XTT 7 M-48- M-48- M-48- M-48- M-48- M-48- M-48- M-48- WST WST WST WST WST WST WST WST 8 P-48- P-48- P-48- P-48- P-48- P-48- P-48- P-48- WST WST WST WST WST WST WST WST 9 R0.01- R0.01- R0.01- R0.01- R0.01- R0.01- R0.01- R0.01- 48-WST 48-WST 48-WST 48-WST 48-WST 48-WST 48-WST 48-WST 10 R0.04- R0.04- R0.04- R0.04- R0.04- R0.04- R0.04- R0.04- 48-WST 48-WST 48-WST 48-WST 48-WST 48-WST 48-WST 48-WST 11 R0.2- R0.2- R0.2- R0.2- R0.2- R0.2- R0.2- R0.2- 48-WST 48-WST 48-WST 48-WST 48-WST 48-WST 48-WST 48-WST 12 R1-48- R1-48- R1-48- R1-48- R1-48- R1-48- R1-48- R1-48- WST WST WST WST WST WST WST WST

[0076] In addition, Tables 8 and 9 describe the conditions of Redensyl treatment on the serum free media after the seeding and the stabilization.

TABLE-US-00008 TABLE 8 96Well-3 A B C D E F G H 1 M-24- M-24- M-24- M-24- M-24- M-24- M-24- M-24- XTT XTT XTT XTT XTT XTT XTT XTT 2 P-24- P-24- P-24- P-24- P-24- P-24- P-24- P-24- XTT XTT XTT XTT XTT XTT XTT XTT 3 R0.01- R0.01- R0.01- R0.01- R0.01- R0.01- R0.01- R0.01- 24-XTT 24-XTT 24-XTT 24-XTT 24-XTT 24-XTT 24-XTT 24-XTT 4 R0.04- R0.04- R0.04- R0.04- R0.04- R0.04- R0.04- R0.04- 24-XTT 24-XTT 24-XTT 24-XTT 24-XTT 24-XTT 24-XTT 24-XTT 5 R0.2- R0.2- R0.2- R0.2- R0.2- R0.2- R0.2- R0.2- 24-XTT 24-XTT 24-XTT 24-XTT 24-XTT 24-XTT 24-XTT 24-XTT 6 R1-24- R1-24- R1-24- R1-24- R1-24- R1-24- R1-24- R1-24- XTT XTT XTT XTT XTT XTT XTT XTT 7 M-24- M-24- M-24- M-24- M-24- M-24- M-24- M-24- WST WST WST WST WST WST WST WST 8 P-24- P-24- P-24- P-24- P-24- P-24- P-24- P-24- WST WST WST WST WST WST WST WST 9 R0.01- R0.01- R0.01- R0.01- R0.01- R0.01- R0.01- R0.01- 24-WST 24-WST 24-WST 24-WST 24-WST 24-WST 24-WST 24-WST 10 R0.04- R0.04- R0.04- R0.04- R0.04- R0.04- R0.04- R0.04- 24-WST 24-WST 24-WST 24-WST 24-WST 24-WST 24-WST 24-WST 11 R0.2- R0.2- R0.2- R0.2- R0.2- R0.2- R0.2- R0.2- 24-WST 24-WST 24-WST 24-WST 24-WST 24-WST 24-WST 24-WST 12 R1-24- R1-24- R1-24- R1-24- R1-24- R1-24- R1-24- R1-24- WST WST WST WST WST WST WST WST

TABLE-US-00009 TABLE 9 96Well-4 A B C D E F G H 1 M-48- M-48- M-48- M-48- M-48- M-48- M-48- M-48- XTT XTT XTT XTT XTT XTT XTT XTT 2 P-48- P-48- P-48- P-48- P-48- P-48- P-48- P-48- XTT XTT XTT XTT XTT XTT XTT XTT 3 R0.01- R0.01- R0.01- R0.01- R0.01- R0.01- R0.01- R0.01- 48-XTT 48-XTT 48-XTT 48-XTT 48-XTT 48-XTT 48-XTT 48-XTT 4 R0.04- R0.04- R0.04- R0.04- R0.04- R0.04- R0.04- R0.04- 48-XTT 48-XTT 48-XTT 48-XTT 48-XTT 48-XTT 48-XTT 48-XTT 5 R0.2- R0.2- R0.2- R0.2- R0.2- R0.2- R0.2- R0.2- 48-XTT 48-XTT 48-XTT 48-XTT 48-XTT 48-XTT 48-XTT 48-XTT 6 R1-48- R1-48- R1-48- R1-48- R1-48- R1-48- R1-48- R1-48- XTT XTT XTT XTT XTT XTT XTT XTT 7 M-48- M-48- M-48- M-48- M-48- M-48- M-48- M-48- WST WST WST WST WST WST WST WST 8 P-48- P-48- P-48- P-48- P-48- P-48- P-48- P-48- WST WST WST WST WST WST WST WST 9 R0.01- R0.01- R0.01- R0.01- R0.01- R0.01- R0.01- R0.01- 48-WST 48-WST 48-WST 48-WST 48-WST 48-WST 48-WST 48-WST 10 R0.04- R0.04- R0.04- R0.04- R0.04- R0.04- R0.04- R0.04- 48-WST 48-WST 48-WST 48-WST 48-WST 48-WST 48-WST 48-WST 11 R0.2- R0.2- R0.2- R0.2- R0.2- R0.2- R0.2- R0.2- 48-WST 48-WST 48-WST 48-WST 48-WST 48-WST 48-WST 48-WST 12 R1-48- R1-48- R1-48- R1-48- R1-48- R1-48- R1-48- R1-48- WST WST WST WST WST WST WST WST

[0077] Also, Table 10 describes the experimental conditions for irradiating the ultrasound while varying the pressure and the duty percentage of the ultrasound.

TABLE-US-00010 TABLE 10 1 2 3 6Well-1 A 1 MPa, 3% 1 MPa, 2% 1 MPa, 1% B 0.5 MPa, 3% 0.5 MPa, 2% 0.5 MPa, 1% 6Well-2 A 1 MPa, 3% 1 MPa, 2% 1 MPa, 1% B 0.5 MPa, 3% 0.5 MPa, 2% 0.5 MPa, 1% 6Well-3 A 1 MPa, 3% 1 MPa, 2% 1 MPa, 1% B 0.5 MPa, 3% 0.5 MPa, 2% 0.5 MPa, 1% 6Well-4 A Control Control Control B 1.5 MPa, 10% 1.5 MPa, 5% 6Well-5 A B 6Well-6 A B

[0078] By referring to the Table 10, while varying the pressure (0.5 MPa, 1 MPa, and 1.5 MPa) and the duty percentage (1%, 2%, and 3%) of the ultrasound, the experiment was performed in which the cells were irradiated with the ultrasound for 10 minutes.

[0079] Specifically, in the wells No. 1 to No. 3, the ultrasound with the pressure of 0.5 MPa or 1 MPa, and the duty percentage of 1% to 3% is used.

[0080] However, in the well No. 4, the higher pressure (1.5 MPa) and the higher duty percentage (5% and 10%) were used for inducing cell death in order to compare the results of other pressures and other duty percentages with the results thereof.

[0081] The results are shown in FIGS. 5 to 21.

[0082] First, FIG. 5 shows the viability of the human outer root sheath cells at a specific point of time and the viability of the human outer root sheath cells after 12 hours from the specific point of time, wherein the human outer root sheath cells are not irradiated with the ultrasound, serving as the control group to be used for comparison with an experiment group with which the ultrasound is irradiated.

[0083] By referring to FIG. 5, if the human outer root sheath cells are not irradiated with the ultrasound then no significant changes are observed in the viability after 12 hours from the specific point of time represented in drawings shown on a right side, compared to the viability at the specific point of time represented in drawings shown on a left side.

[0084] FIG. 6 is a drawing schematically illustrating experimental results right after the human outer root sheath cells are irradiated with the ultrasound (the pressure: 0.5 MPa and the duty percentage: 1%) in accordance with one example embodiment of the present disclosure.

[0085] FIG. 7 is a drawing schematically illustrating experimental results 12 hours after the human outer root sheath cells are irradiated with the ultrasound (the pressure: 0.5 MPa and the duty percentage: 1%) in accordance with one example embodiment of the present disclosure.

[0086] By referring to FIGS. 6 and 7, compared to the results of the control group after 12 hours, an increase is observed in the viability of the cells 12 hours after the cells are irradiated with the ultrasound (the pressure: 0.5 MPa and the duty percentage: 1%).

[0087] FIG. 8 is a drawing schematically illustrating experimental results right after the human outer root sheath cells are irradiated with the ultrasound (the pressure: 0.5 MPa and the duty percentage: 2%) in accordance with one example embodiment of the present disclosure.

[0088] FIG. 9 is a drawing schematically illustrating experimental results 12 hours after the human outer root sheath cells are irradiated with the ultrasound (the pressure: 0.5 MPa and the duty percentage: 2%) in accordance with one example embodiment of the present disclosure.

[0089] By referring to FIGS. 8 and 9, compared to the results of the control group after 12 hours, an increase is observed in the viability of the cells 12 hours after the cells are irradiated with the ultrasound (the pressure: 0.5 MPa and the duty percentage: 2%).

[0090] FIG. 10 is a drawing schematically illustrating experimental results right after the human outer root sheath cells are irradiated with the ultrasound (the pressure: 0.5 MPa and the duty percentage: 3%) in accordance with one example embodiment of the present disclosure.

[0091] FIG. 11 is a drawing schematically illustrating experimental results 12 hours after the human outer root sheath cells are irradiated with the ultrasound (the pressure: 0.5 MPa and the duty percentage: 3%) in accordance with one example embodiment of the present disclosure.

[0092] By referring to FIGS. 10 and 11, compared to the results of the control group after 12 hours, an increase is observed in the viability of the cells 12 hours after the cells are irradiated with the ultrasound (the pressure: 0.5 MPa and the duty percentage: 3%).

[0093] FIG. 12 is a drawing schematically illustrating experimental results right after the human outer root sheath cells are irradiated with the ultrasound (the pressure: 1 MPa and the duty percentage: 1%) in accordance with one example embodiment of the present disclosure.

[0094] FIG. 13 is a drawing schematically illustrating experimental results 12 hours after the human outer root sheath cells are irradiated with the ultrasound (the pressure: 1 MPa and the duty percentage: 1%) in accordance with one example embodiment of the present disclosure.

[0095] By referring to FIGS. 12 and 13, compared to the results of the control group after 12 hours, an increase is observed in the viability of the cells 12 hours after the cells are irradiated with the ultrasound (the pressure: 1 MPa and the duty percentage: 1%).

[0096] FIG. 14 is a drawing schematically illustrating experimental results right after the human outer root sheath cells are irradiated with the ultrasound (the pressure: 1 MPa and the duty percentage: 2%) in accordance with one example embodiment of the present disclosure.

[0097] FIG. 15 is a drawing schematically illustrating experimental results 12 hours after the human outer root sheath cells are irradiated with the ultrasound (the pressure: 1 MPa and the duty percentage: 2%) in accordance with one example embodiment of the present disclosure.

[0098] By referring to FIGS. 14 and 15, compared to the results of the control group after 12 hours, a decrease is observed in the viability of the cells, for example, cell bursting or deformation, 12 hours after the cells are irradiated with the ultrasound (the pressure: 1 MPa and the duty percentage: 2%).

[0099] FIG. 16 is a drawing schematically illustrating experimental results right after the human outer root sheath cells are irradiated with the ultrasound (the pressure: 1 MPa and the duty percentage: 3%) in accordance with one example embodiment of the present disclosure.

[0100] FIG. 17 is a drawing schematically illustrating experimental results 12 hours after the human outer root sheath cells are irradiated with the ultrasound (the pressure: 1 MPa and the duty percentage: 3%) in accordance with one example embodiment of the present disclosure.

[0101] By referring to FIGS. 16 and 17, compared to the results of the control group after 12 hours, a decrease is observed in the viability of the cells, for example, the cell bursting or the deformation, at the time of the ultrasound irradiation and 12 hours after the ultrasound irradiation (the pressure: 1 MPa and the duty percentage: 3%).

[0102] FIG. 18 is a drawing schematically illustrating experimental results right after the human outer root sheath cells are irradiated with the ultrasound (the pressure: 1.5 MPa and the duty percentage: 5%) in accordance with one example embodiment of the present disclosure.

[0103] FIG. 19 is a drawing schematically illustrating experimental results 12 hours after the human outer root sheath cells are irradiated with the ultrasound (the pressure: 1.5 MPa and the duty percentage: 5%) in accordance with one example embodiment of the present disclosure.

[0104] By referring to FIGS. 18 and 19, compared to the results of the control group after 12 hours, a decrease is observed in the viability of the cells, for example, the cell bursting or the deformation with a high ratio, at the time of the ultrasound irradiation and 12 hours after the ultrasound irradiation (the pressure: 1.5 MPa and the duty percentage: 5%).

[0105] FIG. 20 is a drawing schematically illustrating experimental results right after the human outer root sheath cells are irradiated with the ultrasound (the pressure: 1.5 MPa and the duty percentage: 10%) in accordance with one example embodiment of the present disclosure.

[0106] FIG. 21 is a drawing schematically illustrating experimental results 12 hours after the human outer root sheath cells are irradiated with the ultrasound (the pressure: 1.5 MPa and the duty percentage: 10%) in accordance with one example embodiment of the present disclosure.

[0107] By referring to FIGS. 20 and 21, compared to the results of the control group, a decrease is observed in the viability of the cells, for example, the cell bursting or the deformation of most of the cells, at the time of the ultrasound irradiation and 12 hours after the ultrasound irradiation (the pressure: 1.5 MPa and the duty percentage: 10%).

[0108] As such, by referring to FIGS. 6 to 21, the ultrasound of the pressure of 1 MPa or less, the duty percentage of 5% or less, and the intensity of 416.7 mW/cm.sup.2 or less is confirmed to be safe for the cells.

[0109] In addition, when the pressure was set to 1.5 MPa or more, the cell bursting or the deformation is observed. Also, compared to the control group, an increase in growth of the cells is observed when the cells are irradiated with the ultrasound in a safe energy range.

[0110] As an example, in accordance with the method for increasing the viability of the cells by irradiating the cells with the ultrasound, on condition that the drug and the epidermal drug carrier are applied on epidermis of a subject and that the ultrasound parameters have been preset within respective ranges, the ultrasound irradiating device may position an ultrasonic transducer within a threshold range from the epidermis of the subject and then may irradiate the epidermis with the ultrasound. Herein, the epidermal drug carrier may cause cavitation to create at least one cavity around the epidermis in response to irradiating the epidermis with the ultrasound. Further, the ultrasound parameters may include the pressure of the ultrasound and the duty percentage of the ultrasound. And the pressure of the ultrasound may range from 0.5 MPa to 1 MPa and the duty percentage of the ultrasound may range from 1% to 5%.

[0111] Also, the ultrasound parameters may further include the intensity of the ultrasound, which may be in a range from 166.7 mW/cm.sup.2 to 416.7 mW/cm.sup.2.

[0112] Also, the ultrasound parameters may further include a total time of the ultrasound irradiation which may be ten minutes, but the scope of the present disclosure is not limited thereto and the total time of the ultrasound irradiation may be shorter or longer than ten minutes.

[0113] In addition, the ultrasonic transducer may irradiate the epidermis of the subject with the ultrasound, in contact with the epidermis or at a certain distance from the epidermis. In addition, the ultrasonic transducer may have a shape surrounding the epidermis of the subject in a form of a helmet or a headgear.

[0114] Meanwhile, the ultrasound parameters may further include the frequency of the ultrasound which may range from 0.5 MHz to 4.6 MHz.

[0115] As an example, the frequency of the ultrasound may be 1 MHz.

[0116] Meanwhile, the drug may be Redensyl and a concentration of the Redensyl may range from 0.5% to 1%.

[0117] Also, the cells may be the outer root sheath cells.

[0118] FIGS. 22 to 25 are drawings schematically illustrating (i) experimental results of the viability of the human outer root sheath cells derived by only applying the drug of various concentrations and (ii) experimental results of the viability of the human outer root sheath cells derived by applying the drug of the various concentrations together with the epidermal drug carrier and the ultrasound.

[0119] As an example, as can be seen in FIG. 22, under a condition of applying the ultrasound of a same intensity (0.5 MPa, 2%), the highest cell viability is observed when the concentration of the drug is 0.5% to 1%.

[0120] In addition, when the ultrasound of an appropriate intensity, for example, the ultrasound with the pressure of 0.5 MPa and the duty percentage of 2% is applied together with the drug (0.5% to 1% concentration) and the epidermal drug carrier, a significantly higher viability of the cells is observed compared to a result of only applying the drug of the same concentration.

[0121] Meanwhile, since description of FIG. 22 is similar to that of FIGS. 23 and 24, detailed description is omitted.

[0122] Also, as shown in FIGS. 22 to 24, when the intensity of the ultrasound is 166.7 mW/cm.sup.2 (0.5 MPa, 2%), 333.4 mW/cm.sup.2 (1 MPa, 1%), and 416.7 mW/cm.sup.2 (0.5 MPa, 5%), it is observed that the viability of the cells is significantly higher than that in other ranges of the intensity, for example, as shown in FIG. 25, when the pressure of the ultrasound is 1 MPa and the duty percentage is 3%.

[0123] For reference, Tables 11 to 14 below describe the experimental results as shown in FIGS. 22 to 25 respectively.

TABLE-US-00011 TABLE 11 av sd Control 100% 0.00 0.5 MPa 2% 124% 0.03 Drug only Redensyl 0.04% 113% 0.02 Redensyl 0.1% 114% 0.01 Redensyl 0.2% 118% 0.04 Redensyl 0.5% 119% 0.04 Redensyl 1% 123% 0.01 Redensyl 2% 125% 0.01 Redensyl 3% 132% 0.01 Ultrasound Redensyl 0.04% 117% 0.04 0.5 MPa 2% + Redensyl 0.1% 121% 0.05 epidermal drug Redensyl 0.2% 112% 0.03 carrier + drug Redensyl 0.5% 144% 0.06 Redensyl 1% 144% 0.02 Redensyl 2% 128% 0.02 Redensyl 3% 106% 0.07

TABLE-US-00012 TABLE 12 av sd Control 100% 0.00 0.5 MPa 5% 125% 0.01 Drug only Redensyl 0.04% 113% 0.02 Redensyl 0.1% 114% 0.01 Redensyl 0.2% 118% 0.04 Redensyl 0.5% 119% 0.04 Redensyl 1% 123% 0.01 Redensyl 2% 125% 0.01 Redensyl 3% 132% 0.01 Ultrasound Redensyl 0.04% 127% 0.03 0.5 MPa 5% + Redensyl 0.1% 127% 0.04 epidermal drug Redensyl 0.2% 128% 0.09 carrier + drug Redensyl 0.5% 148% 0.04 Redensyl 1% 125% 0.03 Redensyl 2%  99% 0.05 Redensyl 3%  88% 0.01

TABLE-US-00013 TABLE 13 av sd Control 100% 0.00 1 MPa 1% 118% 0.02 Drug only Redensyl 0.04% 113% 0.02 Redensyl 0.1% 114% 0.01 Redensyl 0.2% 118% 0.04 Redensyl 0.5% 119% 0.04 Redensyl 1% 123% 0.01 Redensyl 2% 125% 0.01 Redensyl 3% 132% 0.01 Ultrasound Redensyl 0.04%  97% 0.02 1 MPa 1% + Redensyl 0.1% 107% 0.01 epidermal drug Redensyl 0.2% 110% 0.02 carrier + drug Redensyl 0.5% 143% 0.17 Redensyl 1% 134% 0.01 Redensyl 2% 114% 0.03 Redensyl 3% 104% 0.00

TABLE-US-00014 TABLE 14 av sd Control 100% 0.00 1 MPa 3%  32% 0.05 Drug only Redensyl 0.04% 113% 0.02 Redensyl 0.1% 114% 0.01 Redensyl 0.2% 118% 0.04 Redensyl 0.5% 119% 0.04 Redensyl 1% 123% 0.01 Redensyl 2% 125% 0.01 Redensyl 3% 132% 0.01 Ultrasound Redensyl 0.04%  24% 0.01 1 MPa 3% + Redensyl 0.1%  20% 0.02 epidermal drug Redensyl 0.2%  20% 0.00 carrier + drug Redensyl 0.5%  17% 0.02 Redensyl 1%  25% 0.02 Redensyl 2%  13% 0.01 Redensyl 3%  6% 0.01

[0124] Meanwhile, FIGS. 26 to 37 are drawings schematically illustrating experimental results of gene expression in response to irradiating the cells with the ultrasound in accordance with one example embodiment of the present disclosure.

[0125] Specifically, FIGS. 26 to 37 are drawings schematically illustrating the experimental results of the gene expression related to WNT10B, Beta Catenin, Keratin 15, VDR, PCNA, Ki67 and BCL2 when the drugs are applied to the epidermis of the subject and the epidermis is irradiated with the ultrasound having the frequency of 1 MHz.

[0126] As an example, by referring to FIGS. 26 to 29, when the intensity of the ultrasound is set to 166.7 mW/cm.sup.2 (0.5 MPa, 2%), the frequency of the ultrasound is set to 1 MHz, and the concentration of Redensyl is at 0.5% to 1%, it is observed that the gene expression levels related to WNT10B, Beta Catenin, Keratin 15, VDR, PCNA, Ki67 and BCL2 are significantly higher than those in other ranges of the ultrasound.

[0127] FIGS. 30 to 33 show a case where the intensity of the ultrasound is set to 416.7 mW/cm.sup.2 (0.5 MPa, 5%), the frequency of the ultrasound is set to 1 MHz. And similar to description of FIGS. 26 to 29, it is observed that the gene expression levels related to WNT10B, Beta Catenin, Keratin 15, VDR, PCNA, Ki67 and BCL2 are significantly higher in a range, where the concentration of Redensyl is at 0.5% to 1%, than those in other ranges of the ultrasound.

[0128] FIGS. 34 to 37 show a case where the intensity of the ultrasound is set to 333.4 mW/cm.sup.2 (1 MPa, 1%), the frequency of the ultrasound is set to 1 MHz. And similar to description of FIGS. 26 to 29, it is observed that the gene expression levels related to WNT10B, Beta Catenin, Keratin 15, VDR, PCNA, Ki67 and BCL2 are significantly higher in a range, where the concentration of Redensyl is at 0.5% to 1%, than those in other ranges of the ultrasound.

[0129] Meanwhile, FIGS. 38 and 39 are drawings schematically illustrating degrees of DNA methylation in response to irradiating the cells with the ultrasound having the frequency of 1 MHz in accordance with one example embodiment of the present disclosure.

[0130] As can be seen by referring to FIGS. 38 and 39, when the ultrasound with the frequency of 1 MHz and the intensity of 166.7 mW/cm.sup.2 to 416.7 mW/cm.sup.2 is applied together with the drug having a concentration of 0.5% to 1% and the epidermal drug carrier, it is observed that the unmethylated DNA Ct values were significantly lowered.

[0131] Meanwhile, the ultrasound parameters may further include PRF or PRP. Herein, PRF may range from 1 Hz to 100 Hz and PRP may range from 0.01 second to 1 second.

[0132] Meanwhile, FIG. 42 is a drawing schematically illustrating clinical results according to a lapse of time when the epidermal drug carrier, the drug, and the ultrasound are applied together for 10 minutes two times a week in accordance with one example embodiment of the present disclosure.

[0133] FIG. 43A is a drawing schematically illustrating a case where no treatment is applied. FIG. 43B is a drawing schematically illustrating a case where a conventional treatment is applied for 12 months. FIGS. 43C and 43D are drawings schematically illustrating clinical results according to a lapse of time when the epidermal drug carrier, the drug, and the ultrasound are applied together in accordance with one example embodiment of the present disclosure.

[0134] As compared with FIG. 43A, improved results can be confirmed also in FIG. 43B. Further, in FIG. 43B, when compared with FIGS. 43C and 43D, it is observed that there is a very large difference in a time of application. That is, in accordance with the present disclosure, a result same as or superior to that of conventional techniques is acquired within the time of the application (2 to 4 months in case of FIGS. 43C and 43D) remarkably shorter than that (12 months in case of FIG. 43B) of the conventional techniques.

[0135] FIGS. 44A and 44B are drawings schematically illustrating clinical results according to a lapse of time before and after the epidermal drug carrier, the drug, and the ultrasound are applied together for 6 months in accordance with one example embodiment of the present disclosure.

[0136] Even with naked eye, the difference between before and after the application can be seen in the number of hairs, etc.

[0137] The present disclosure has an effect of increasing the viability of the cells in a non-invasive and painless manner by irradiating the cells with the ultrasound.

[0138] The present disclosure has another effect of further increasing the viability of the cells by using the drug together with the epidermal drug carrier and the ultrasound compared to a case of only using the drug with a same concentration.

[0139] As seen above, the present disclosure has been explained by specific matters such as detailed components, limited embodiments, and drawings. They have been provided only to help more general understanding of the present disclosure. It, however, will be understood by those skilled in the art that various changes and modification may be made from the description without departing from the spirit and scope of the disclosure as defined in the following claims.

[0140] Accordingly, the spirit of the present disclosure must not be confined to the explained embodiments, and the following patent claims as well as everything including variations equal or equivalent to the patent claims pertain to the category of the spirit of the present disclosure.