A novel anti-aging skincare cream, the cosmetic device and cream within
Technical Field
The present invention relates to a novel anti-aging skincare cream, the cosmetic device and cream within to stimulate collagen synthesis, and more specifically, to a method for stimulating collagen synthesis using programmed LED light in combination with the PLA peptide.
Background Art
The most visible signs of aging are exemplified in the skin. Aged skin exhibits wrinkles, lacks elasticity, and loses radiance. The biological core to the skin aging is that the levels of collagen protein synthesized in the skin gradually decrease over time. The younger we are, the more collagen our skin produces; the older we are, the less collagen our skin maintains. It is therefore the challenge to develop methods to maintain high levels of collagen in the skin, in order to achieve the effects of skin rejuvenation. The collagen protein in the skin is dynamically regulated (illustrated in Figure 1). First, collagen is regulated at the production level. Fibroblasts in the dermis layer of the skin are responsible for the collagen production. In fibroblasts, the gene encoding pro-collagen, the collagen precursor, is turned on in the nucleus to generate the messengers, which are transported to the cytoplasm and used as a template to generate the pro-collagen protein. The newly synthesized pro-collagen protein is processed to become mature collagen protein and exported out of fibroblasts to be integrated into the extracellular matrix network of the skin. This genetic mechanism of collagen production is fundamental to our skin. It is regulated by our biological age and our environmental stimuli. Since we cannot reverse our biological age, we have developed numerous tools to stimulate this genetic pathway to produce more collagen proteins. These tools include chemical intervention, such as retinoic acid, and physical intervention, such as dermabrasion. Almost all of the tools in the market directly or indirectly manipulate this collagen production pathway to achieve high levels of collagen, thus relieving the aging skin defects.
However, collagen proteins produced by the abovementioned stimulation would not be maintained in the skin. In fact, our skin has developed several natural feedback response mechanisms to remove the excessive new collagen deposited to the matrix so as to bring the level of skin collagen to match the physiological skin environment defined by our age. One most efficient mechanism is to degrade the collagens by activating the collagen-degrading enzyme collagenases, or matix metalloproteinases (MMPs). Studies have shown that collagenases are
also responsible for the turnover of the natural amounts of collagen the fibrobalsts continuously produce. During the aging process, collagenase activity increases to accelerate the degradation of collagen. The increased degradation of collagen, coupled with the decreased production, accounts for the age-dependent loss of total collagen in the skin. No attempts have been developed to address the feedback regulation of collagen levels, explaining the temporary relief of skin aging signs by the available means developed so far.
Therefore, there are ample rooms for developing new tools to target the regulatory turnover of collagen in the skin, particularly to intervene the feedback mechanism to remove newly synthesized collagen. Combinatorial methods to stimulate the genetic pathway to produce more new collagen into the matrix and to prevent the collagenase-mediated degradation of new collagen would in principle be able to accumulate high collagen levels in the skin to achieve the ultimate goal of skin rejuvenation.
Disclosure of Invention According to our understanding of the biology of dynamic collagen regulation in the skin, the inventors have invented a method to address the maintenance and production of new collagen, either alone or in combination.
In one aspect, the present invention relates to a peptide with the following general formula I: X.sup.1 -X.sup.2 -X.sup.3 -NHOH ( formula I ) wherein X.sup.1 is a residue of an amino acid selected from L or D form of amino acid selected from the group consisting of proline, hydroxyproline, thioproline and alanine, X.sup.2 is a residue of an amino acid selected from L or D form of amino acid selected from the group consisting of glutamine, glutamic acid, leucine, isoleucine and phenylalanine and X.sup.3 is a residue of an amino acid selected from L or D form of amino acid selected from the group consisting of glycine, alanine, valine, leucine and sarcosine; and wherein the carboxyl group of .alpha. -amino acid X.sup.1 forms a peptide bond together with the amino group of . alpha. - amino acid X.sup.2, the carboxyl group of .alpha.-amino acid and acid X.sup.2 forms a peptide bond together with the amino group of .alpha.-amino acid X.sup.3, and the carboxyl group of .alpha.- amino acid X.sup.3 forms an amido together with --NH0H; and the hydrogen atom of the amino group in said .alpha.-amino acids X.sup.1 may be replaced with a radical selected from the group consisting of acetyl, benzoyl, benzyloxy, t-butyloxycarbonyl, benzyloxycarbonyl, p- aminobenzoyl, p-amino-benzyl and p-hydroxybenzoyl; or a pharmaceutically acceptable salt thereof.
Preferably, the peptide has the sequence of Benzoyl-Proline-D-Leucine-D-Alanine-NHOH.
In one further aspect, the present invention relates to a skin-care cream which contains the above mentioned peptide and/ or any acceptable skin-care cream additives.
In one further aspect, the present invention relates to a use of the peptide of formula I in the preparation of skin- care cream. In still one further aspect, the present invention relates to the use of the peptide in the preparation of cream to reduce wrinkle.
In still one further aspect, the present invention relates to skin-care device comprising of a series of LED bulbs, which are controlled to emit mono-color light of a specific combination of defined wavelength, frequency and intensity. Preferably, the said LED wavelength is 590 nm to 660 nm; preferably 630-660nm, most preferably 660nm; and the said frequency is 1 Hz - 50 Hz, preferably 5-1 OHz; and the said intensity is 50 μW -50 mW per cm2 of illuminated skin area, preferably 1800Lux for 5 minutes or 550Lux for 1 minute.
Preferably, said device has 100-3000 LED bulbs, which are evenly arranged on a plain or curvature surface, and the surface is adjustable to enable each LED is roughly of the same distance from skin surface illuminated.
The device may have a central controller that controls the LED intensity and frequency.
In still one further aspect, the present invention relates to skin caring method comprising the combined applications of the above mentioned cream and the device.
Description of the figures
Fig. 1 illustrates Regulatory pathways for collagen production and maintenance in the skin. The targets of this invention are indicated.
Fig. 2 illustrates the absorbance scanning of cultured human skin fibroblasts reveal absorbance at specific wavelengths. Only a fraction of the scanned wavelength is shown.
Fig. 3 illustrates the custom-made LED circular panels, with adjustable intensity, pulsing frequency and time of illumination, were used to investigate the effects of different light programs on new collagen production in cultured human skin fibroblasts.
Fig. 4 illustrates the programmed LED light at wavelength 660 and the specific collagenase inhibitor synergistically induce production of type I pro-collagen. Actin protein is used as total protein loading control. C: control, no LED light, no inhibitor; RH5 - red LED light alone for 5 minutes; PLA --specific collagenase inhibitor alone; RH5 +PLA: red LED light for 5 minutes first, then addition of specific collagenase inhibitor.
Fig. 5 illustrates the design of LED goggle and operation control.
Fig. 6 illustrates the pictures of the prototype product of the LED goggle and operation control. Fig 6a: frontal view. Fig 6b: rear view.
Fig. 7 illustrates the test subject's result after 4 weeks' of application, once per week LED goggle use (5 minutes each time), and twice a day (once morning and once evening) of collagenase inhibitor cream use.
Fig. 8 illustrates the Clinical Test Results with PLA creams: Subjective Impressions
- PLA cream products (day cream, night cream, eye cream) used daily for 4 weeks
- 70% of participants reported they would purchase the products. Subjects responses: 1) Improved skin dryness
2) Improved skin moisturization
3) Improved firmness and elasticity
4) Reduced under-eye puffiness
5) Improved skin radiance 6) Improved appearance of fine lines
7) Improved skin smoothness.
Fig. 9 illustrates the clinical Test Results: Expert Evaluator Assessment PLA eye system is clinically shown to reduce appearance of fine lines after 4 weeks, as assessed by expert evaluator (once weekly LED goggle use + daily use PLA eye cream. Subjects responses:
1) Firmer skin in the eye area
2) Fewer wrinkles in the eye area
3) Visible reduction in "crow's feet"
4) Visible reduction in the length, depth, and number of lines and wrinkles in the eye area 5) Visible reduction in lines & deep wrinkles both immediately and cumulatively
6) Visible reduction in eye puffiness and bags
7) Dramatic improvement in deeper lines and wrinkles, crows feet, under-eye bags, puffiness, and dark circles
8) Agreed that eye product delivered remarkable improvement in signs of aging in the eye area
9) Dark circles are dramatically reduced. Fig. 10 illustrates HPLC result of the PLA.
Detailed description of the invention
The present invention relates to new peptidylhydroxamic acid derivatives representing by the following formula I that specifically inhibit the activity of collagenases of vertebrate origin, as well as to collagenase inhibitors containing these new peptidylhydroxamic acid derivatives as active ingredients. The indications of these new peptides are used in topical cream to reduce wrinkles and increase skin elasticity and firmness.
A peptidylhydroxamic acid derivative of the general formula: X.sup.l -X.sup.2 -X.sup.3 -NHOH formula I wherein X. sup.1 is a residue of an amino acid selected from L or D form of amino acid selected from the group consisting of proline, hydroxyproline, thioproline and alanine,
X.sup.2 is a residue of an amino acid selected from L or D form of amino acid selected from the group consisting of glutamine, glutamic acid, leucine, isoleucine and phenylalanine and X.sup.3 is a residue of an amino acid selected from L or D form of amino acid selected from the group consisting of glycine, alanine, valine, leucine and sarcosine; and the carboxyl group of .alpha. -amino acid X. sup.1 forms a peptide bond together with the amino group of . alpha. - amino acid X.sup.2, the carboxyl group of .alpha.-amino acid and acid X.sup.2 forms a peptide bond together with the amino group of .alpha.-amino acid X.sup.3, and the carboxyl group of .alpha.- amino acid X.sup.3 forms an amido together with --NH0H; and the hydrogen atom of the amino group in said .alpha.-amino acids X.sup.l may be replaced with a member selected from the group consisting of acetyl, benzoyl, benzyloxy, t- butyloxycarbonyl, benzyloxycarbonyl, p-aminobenzoyl, p-amino-benzyl, p-hydroxybenzoyl or a pharmaceutically acceptable salt thereof.
The present invention relates to an LED (light-emitting diode)-based stimulation of collagen producing method. These small light bulbs are not picked randomly, but their parameters determined using accurate measurement. For instance, the working wavelengths were determined by exposing human fibroblast to an absorbance scanning across the wavelength from 300 to 800 nm. We have identified the absorbance peaked at three distinct wavelengths, 590, 630 and 660 nm (Figure 2). The working intensity and pulsing frequency were determined by using custom-made LED device to stimulate collagen production from cultured human fibroblasts (Figure 3).
Based on extensive research, we have identified some specific combinations of LED parameters that can stimulate new collagen production. We then apply these parameters to the assembly of a novel LED light device, which is made up of variable number of LED bulbs of specific wavelength, specific light emitting angle, specific light strength, and specific emitting
frequency. The final light output is less than 10 mw/cm2 skin area covered. The mild light does not hurt the skin, yet LED light can still penetrate into the skin to stimulate collagen production.
Rigorous testing showed that these programmed LED light can achieve the maximum effects on increasing collagen production when used together with the abovementioned PLA peptide. The scientific principle behind this is that though the specially programmed LED red light that can reach and activate fibroblast cells deep in human skin and induce collagen production, increased collagen concentration in turn increases collagenase activity that would degrade the extra collagen. Our PLA peptide specifically inhibits collagenase, and thus achieves a high steady level of collagen in the skin, either alone or in combination with the LED stimulation (Figure 4).
Extra caution has been exercised when the LED bulbs are aligned in a curved panel for human facial skin treatment. They were spaced evenly and every light bulb was adjusted to fit the curvature of human facial skin. The product and color design was carried out and one simulation sample is shown in Figure 5. Prototype moulds were manufactured and used to produce the first batch of products. Pictures of these products are shown in Figure 6. Human trials of these LED goggles have confirmed their safety to use and effectiveness in skin rejuvenation (See experimental section below).
In summary, this is the first invention that has combined specific wavelength and specific pulse LED light (technical parameter specified below) and a specific collagenase inhibitor cream in a simple goggle model. In our quantitative human skin cell experiments, we have observed significant higher levels of Type I procollagen stimulation (10X) using our specific collagenase inhibitor cream alone, and synergistic procollagen boosting effects (50X) using our LED light and our specific collagenase inhibitor, compared with untreated cells. In our human trials of the light and cream, we observed more than 75% improvement in wrinkle diminishing, eye bag shrinkage, and dark circle elimination after 4 weeks of usage, once a week for 5 minutes using specific LED goggle and twice per day (once morning and once night) using our collagenase inhibitor cream. Even the PLA peptide alone produced great results in skin treatment in human trials (Figures 8 and 9).
Examples
Example 1.
PLA Manufacturing Method
The PLA peptide can be produced by chemical synthesis with three individual amino acids, in combination with end modification. One of such synthesis route can be:
Step 1: CBZ-Cl + H-Pro-OH — -> Z-Pro
Step 2: Boc-D-Leu + D-Ala-OMe.HCL— > Boc-D-Leu-D-Ala-OMe Step 3: Boc-D-Leu-D-Ala-OMe— >D-Leu-D-Ala-OMe + Z-Pro Step 4: D-Leu-D-Ala-OMe + Z-Pro— > Z-Pro-D-Leu-D-Ala-OMe Step 5: Z-Pro-D-Leu-D-Ala-OMe— >Z-Pro-D-Leu-D-Ala Step 6: Z-Pro-D-Leu-D-Ala— >Z-Pro-D-Leu-D-Ala-NHOH. All the reagents are analytical grade and purchased commercially. The final purified peptide is more than 98% pure, as determined by HPLC. One of such quality control analysis is found in Fig. 10.
Example 2.
One example of facial cream formulation with PLA as an active ingredient:
Description (INCI Name) Formula Fucntion
1 Water Vehicle
2 Cyclop entasiloxane Emollient
3 Glycerin Humectant
4 Sodium Hyaluronate(l%) Moisturizer
5 Polysilicone-11 Thickener
6 Theobroma Cacao (Cocoa) Extract Extract
7 Phenoxythanol Preservative
8 Polyacrylamide Thickener
9 Carbomer Thickener
10 Triethanolamine Nuetralizing Agent
11 Polysorbate 20 Solubilizer
12 Dis odium EDTA Chelating Agent
13 Ascorbic Acid Vitamin
14 Polymethylsilsesquioxane Asthetic Modifier
15 C 13- 14 Isoparaffin Solvent
16 Panthenol Vitamin
17 Retinyl Palmitate Vitamin
18 Bis-PEG/PPG 14/14/Dimethicone Wetting Agent
19 Laureth-7 Co-emulsifier
20 Sambucus Nigra Flower Extract Extract
21 Panax Ginseng Root Extract Extract
22 Equisetum Arvense Leaf Extract Extract
23 Calendula Officinalis Flower Extract Extract
24 Salvia Officinalis (Sage) Leaf Extract Extract
25 Geranium Maculatum Extract Extract
26 Honey Extract Extract
27 Butylene Glycol Humectant
28 Sodium PCA Moisturizer
29 Hydroxyapatite Molecular Sieve
30 PLA Peptide Collagnease Inhibitor
Example 3.
Quantitative Skin Cell Experiment with the LED goggle and peptide PLA Skin fibroblast cells (ATCC No.: CRL-1762) were grown in Dulbecco's modified Eagle medium
(DMEM) with additional nutrient 200 mM L-Glutamine, 50 μg/ml gentamycin, and 10% fetal bovine serum. The growth condition was 37 0C with5% CO2. All procedure was conducted in a sterile hood. When skin cells were grown to 50-60% confluence in a plate (10 cm diameter), the media were aspirated, and the skin cells were washed with 5 ml phosphate buffered saline (PBS), then PBS was aspirated. Then 10 ml PBS was added to skin cells in the same plate. Our custom assembled LED round plate was applied to cover the top of the skin cells, and cells were illuminated with programmed LED light at specific wavelength, frequency, and intensity. The LED light was shut off after designated time. After the LED light was turned off, PBS solution in the plate was replaced with 10 ml DMEM media. Our specific collagenase inhibitor at final solution of 1 XlO"5 M was added to the media. The cells were harvested after 12 hours of additional incubation in CO2 incubator. Total protein was extracted and 50 μg of total protein was run on a 5-20% gradient SDS-polyacrylamide gel. Western analysis was performed with Type I procollagen antibody, SP1.D8 from Developmental Studies Hybridoma Bank. The skin cell result is shown in Figure 4.
Example 4 Human trials
Each test subject uses collagenase inhibitor cream twice a day (once in the morning and once in the night) for 4 weeks. Once a week the test subject uses our LED goggle with specification below for 5 minutes (total 4 times). Test result of one typical subject is shown in Figure 7.
LED goggle technical specifications
• Input Voltage: ACl 10V/50-60Hz ±10%
• Output Power: <1 IW
• Output Light Intensity: < 1 OOOLux
• Output Frequency: 4Hz
• LED Numbers: 252
• LED Wavelength: 660 nm
• Pre-set Timer: 5min, 10 min, 20min
• Stop Signal: automatic off with 2 sec buzzing Total Weight: 418.2 grams (Goggle Weight: 222.5 grams)
1. Clinical Test Results with PLA-containing creams only : Objective Measurements PLA cream products (day cream, night cream, eye cream), used daily for 4 weeks 70% of participants reported they would purchase the products Table 1 : Eye Cream (Improvement in area around eye)
Skin Dryness Fine Lines - Wrinkles Under-eye Under-eye Circles
Puffiness
70% 13% 45% 23%
Table 2: Day Cream and Night Cream (Improvement in skin)
Skin Dryness Brightness Elasticity Tone Uniformity of pigmentation Day Cream 70% 10% 23% 14% 28%
Night Cream 69% 14% 26% 13% 31%
5. Test Subjects using both LED goggle and PLA-containing creams respond to questions regarding product performance and benefit
• 100% saw tighter, firmer skin in the eye area;
• 100% agreed that the eye treatment system made them look younger;
• 91% saw fewer wrinkles in the eye area;
• 91% saw a visible, measurable reduction in "crow' s feet" ;
• 91% saw a visible, measurable reduction in the length, depth, and number of lines and wrinkles in the eye area;
• 75% saw a significant visible reduction in lines and deep wrinkles both immediately and cumulatively; • 75% saw a visible reduction in puffmess and under-eye bags;
• 75% saw an overall dramatic improvement in deep defined lines and wrinkles, crows feet, under-eye bags, puffmess, and dark circles;
• 75% agreed that this product delivered dramatic, noticeable improvement in reducing signs of aging in the eye area; • 67% saw dark circles dramatically reduced.
One skilled in the art can understand the above examples are for the purpose of illustration without limiting the scope of the invention.