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Synopsis - Biopeptide-CL
Description: Synthetic peptide in hydroglycolic solution:
100 ppm of Palmitoyl-Gly-His-Lys
CTFA/INCI name: Polyglyceryl Methacrylate (and) Propylene Glycol
(and) Palmitoyl Oligopeptide
Demonstrated cosmetic activity:
* In vitro:
* synthesis of collagen by fibroblasts: +350%
* synthesis of GAG (hyaluronic acid) by fibroblasts: +146%
* Ex vivo (histology of biopsies):
* synthesis of collagen and skin repair: +96% (Retinoic acid: +92%)
* In vivo: effects observed after one month of treatment
* Thickening of the skin: +4% (ultrasound scan)
* Anti-wrinkle effects: roughness -17%
mean depth of wrinkles -23%
depth of main wrinkle (crow's feet) -39%
Use: Anti-wrinkle, anti-aging, eye zone, facial care, facial lift and firming
Recommended concentration: 3-5%
"Wrinkles are nothing more or less than the depletion of collagen in the skin."
Quotation from Professor Robert Garonne
Professor of Cell Biology
Vice-Principal of Lyon University (France)
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INTRODUCTION
Collagen – skin – retinoids
Collagen
Collagen is one of the proteins found in the connective tissue of vertebrates. Its primary structure (amino acid composition), secondary and tertiary structures (spatial conformation, triple helix arrangement) and higher structure (formation of fibrils and fibers, network in the tissue), its biochemistry (synthesis, hydroxylation, degradation and reticulation) and its physiological roles in various organs have described in detail in many publications.
The skin
Like other tissues, the skin is continuously being replaced and its turnover rate decreases with age. The basal cells of the germ cell layer of the epidermis divide to produce cells that then differentiate and migrate to the surface of the skin, where they undergo changes. In the dermis, the fibroblasts synthesize and secrete extra-cellular molecules of collagen to replace and regenerate the connective tissue. During the healing process, after an impact that has damaged the tissue, de novo synthesis of collagen is also important.
The extra-cellular matrix of the dermis also contains other macromolecules than the ubiquitous collagen. The glycoaminoglycans, such as dermatan sulfate, chodroitin sulfate and hyaluronic acid, are involved in the construction of the connective tissue. These substances are also synthesized by the fibroblasts; their synthesis rate declines with age. This deficiency leads to dehydration of the skin, which also becomes thinner and wrinkle formation.
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The Retinoids
The retinoids (derivatives of Vitamin A: retinoic acid, alcohol, aldehyde and esters) have many dermatopharmacological effects that are well documents in the literature.
By way of example, we will cite several key publications:
In 1989, Kligman showed that topical treatment with retinoic acid (all trans) boosts the repair activity of the fibroblasts in the dermis after prolonged exposure to UV radiation. This substance stimulates the in-vivo synthesis of collagen and leads to thickening of the dermis. Some of the photo-aging effects are reversible when retinoic acid is applied.
Bryce et al. (1988) confirmed the effect of retinoic acid in repairing skin damage caused by exposure to UV and showed that his molecule obliterates wrinkles. These authors also observed that exposure to UV results in thickening of the skin, affecting both the dermis and the epidermis. This thickening is increased by treatment with retinoic acid. In the model investigated, the authors attributed the disappearance of wrinkles to the consequence of the restructuring of the collagen matrix: the neo-collagen “squashes” the disorganized elastic fibers, which caused the wrinkled appearance. These morphological studies are supported by biochemical studies which use immunofluorescence to detect the synthesis of collagen induced in vivo by retinoic acid after exposure to UV (Schwartz and Kligman, 1991).
The review article by Gendimenico (1993) summarizes the known effects of retinoic acid on the differentiation of keratinocytes, on the proliferation of fibroblasts and other cell types in the skin (melanocytes and sebocytes). Retinoic acid stimulates the proliferation of keratinocytes, which accounts for the thickening of the skin in response to this treatment. Recent studies also seem to demonstrate that in the dermis, retinoic acid may stimulate the synthesis of elastin, which contributes to the well – established anti-wrinkle effect of this substance.
In contrast, it is known that the use of retinoic acid for cosmetic purposes is prohibited, that it has a high irritation potential and, at high does levels, is teratogenic.
It has recently been discovered that some fragments of native collagen are capable of stimulating the growth of fibroblast-type cells and of inducing them to synthesis and excrete collagen (Macquart et al., 1988). It has therefore been suggested that the degradation of this macromolecule (damage, age) by collagenases results in the releases of one or more factors which initiate the synthesis of fresh collagen.
One of these stimulatory peptide factors is the tripeptide Gly-His-Lys.
The Glycyl-Histidyl-Lysine tripeptide is a fragment of the collagen chain. By cutting a molecule from the alpha (2)1-chain of human collagen, this peptide is released together with a multitude of other fragments. This peptide was initially described as a growth stimulant and then as a factor involved in healing (Simeon et at., 1999)
In vitro experiements have demonstrated that very low concentrations of this peptide (of the order of 10(-9) and 10(-12) Mole/I) are able to induce the synthesis of extra-cellular collagen by the dermal fibroblasts, but without increasing their number.
In the light of what has been said, we thought that it would be interesting to develop a cosmetic active ingredient which:
· is a natural biomimetic derivative (a fragment of collagen),
· has stimulatory activity comparable to that of retinoic acid, but without its drawbacks.
BIOPEPTIDE-CL is the outcome of this research.
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2. DESCRIPTION OF BIOPEPTIDE-CL
We prepared the peptide Palmtoyl-Gly-Lys, which consists of the three natural amino acids, Glycine, Histidine and Lysine, by a peptide synthesis pathway. In order to give this natural peptide lipophilic properties, we attached palmitic acid of plant origin, which has the following structure:
BIOPEPTIDE-CL is a preservative-free solution of this peptide in a fluid hydroglycolic gel. The concentration of the Pal-Gly-His-Lys peptide in this gel is about 100ppm (= 100mg/l). As will be described below, this concentration corresponds to very high peptide activity.
2.1 Bio-active peptides
“Peptides” are molecules that consist of a chain of amino acids, the size of which expressed in terms of molecular weight is generally below 10,000 daltons (about 80-100 amino acids). Above this size, molecules are described as “proteins” rather than “peptides.”
*where n = 1 to 90.
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A very large number of different peptides are found in the animal, plant and microbial kingdoms. These peptides range from a simple linkage of two amino acids (dipeptides) to oligomeric linear or cyclic chains (some antibiotics) and even to complex structures such as insulin and neurotoxins.
The peptides with which are concerned here belong to the domain of the oligopeptides (less than 10 amino acids long), of which the amino acid sequence and biological activity have been fully characterized.
The activities of these peptides are very diverse: they can play roles of messengers, stimulants, enzyme inhibitors and neurotransmitters. They are involved in regulating growth, lactation, sleep and pain, blood pressure, vasoconstriction or vasodilatation, the immune response, the mutual recognition of cells, digestion, healing and many other physiological and metabolic functions.
At the physiological level, peptides are ephemeral substances. They are usually generated as a result of a specific process in which the “precursor proteins” are cut and secreted by specialized cells. Their life span in the target of their action (receptor cells) is usually very short, of the order of a few minutes. Peptidolytic and proteolytic enzymes are responsible for the rapid catabolism of these potent substances, which produce their maximum effect at very low concentrations; most biologically active peptides are reactive at the nano- or micromole scale (a few µg/l).
It is therefore obvious that this group of naturally-occurring substances is of considerable physiological and pharmacological interest. However, their potential for use in cosmetogoly had not previously been thoroughly investigated; it had been assumed that peptides had little chance of penetrating the stratum corneum.
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2.2 Cutaneous diffusion
Most of the oligopeptides described above are highly hydrophilic because of their “zwitterions” or “betain” structure, i.e. the pressure of an NH+3 group and a COO- group within the same molecule.
The functional groups of many amino acid side chains also contribute to this high solubility in water.
Where R:
For these reasons, the use of a bioactive peptide for cosmetic purposes runs into a major natural obstacle: the skin barrier of the stratum corneum.
This lipid shield, which has a water content of no more than 13%, only allows a very select range of endogenous substances to get through. The skin diffusion of a natural peptide into the stratum corneum is therefore a slow and inefficient process due to the opposing natures of the molecules involved (a hydrophilic peptide and a lipophilic barrier).
Consequently, we attempted to modify these peptides which are of cosmetic value. In particular the peptide fragment of collagen Gly-His-Lys was modified in order to confer on it the following characteristics:
· a chemically stable form;
· a marked affinity for the lipids of the skin
· an identified and specific biological activity.
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Comparative “tracer” method study
Carnosine vs. lipocarnosine
Carried out by the Societe de Pharmacologie et d’Immunologie-BIO (SPI-BIO) of the French Atomic Energy Commissariat at Saclay (France) in accordance with Good Laboratory Practice standards.
The principle consists of comparing the cutaneous diffusion response of carnosine (a dipeptide with the sequence ß-Alanyl-Histidine) chosen as a model peptide, with the behavior of palmitoyl-carnosine in the following conditions:
· [125 l] labeling on the histidyl side chain
· application of a solution of the labeled peptide to the epidermal side of skin biopsies
· determination of the radioactivity distributed in the layers of the stratum corneum and the entire epidermis/dermis using a counter.
The solutions containing the radiolabeled peptides (carnosine and palmitoyl-carnosine) were placed on a fragment of skin mounted in a diffusion cell. Samples were taken after 30 minutes, 3 hours and 6 hours: the stratum corneum was investigated in detail by the stripping method. The epidermis/dermis was directly incinerated and analyzed using a gamma counter.
Results
* Uptake and distribution in the skin
The results of the determination of the distribution of [125 l] radioactivity in the various structures of the skin after applying [125 l]- carnosine and [125 l]- lipocarnosine are shown in histogram form Figures 1 and 2 respectively.
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In general the uptake of [125 l] radioactivity after applying [125 l]-carnosine was very low and corresponded to less than 1% of the dose applied, whereas in contrast, after applying [125 l]-lipocarnosine, about 25% of the dose applied was found bound to the skin. The level of uptake was particularly high in the stratum corneum.
The distribution in the stratum corneum is shown in Figure 3. It shows that the first 4 or 5 rows are strongly radiolabeled.
* Diffusion through the skin
Figure 4 shows that only a tiny fraction of the substance applied crosses the skin:<0.05% over six hours.
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* Conclusion
It emerges clearly that the palmitoyl-peptides have two advantages:
·* the fatty acid attached by an amide bond confers very great affinity on the peptide model chosen for the stratum corneum (˜ 100 times greater)
· * and epidermal absorption (beyond the stratum corneum) at least 10 times higher.
However, this preferential uptake by the epidermis is not accompanied by increased transcutaneous penetration because the flow is very limited in both cases (initial and modified peptide).
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3. SUBSTANTIATION TESTS
3.1 In vitro tests
· * In vitro stimulation of collagen synthesis
[study carried out by EVIC CIBA]
The fibroblasts are responsible for producing and maintaining the extracellular matrix (EMC) within the connective tissues.
The purpose of this study was to assess the synthesis of one of the constituents of the ECM, collagen, in the presence of several concentrations of the peptide Pal-Gly-His-Lys (active principle of Biopeptide CL).
* Principle
Determination of the efficacy of the substance by measuring the radioactivity of the molecules of collagen synthesized by the fibroblasts after they had incorporated labeled praline from the culture medium.
Reagents: DMEM culture medium, neutral collagen, tritiated praline (3H-proline), and pepsin.
Strain: primary culture of dermal fibroblasts of human origin.
Experimental protocol: Fibroblasts were seeded on 96-well microplates and then allowed to incubate to total confluence. At confluence, the culture medium was replaced by a medium containing a given concentration of the test substance. After incubating for 24 hours, the wells were emptied and the medium replaced by fresh medium containing a known concentration of the test substance plus labeled praline. Incubation period: 24 hours. The collagen was then extracted by differential centrifugation and centrifuging. The radioactivity of the collagen molecules synthesized by the cells was measured using a Beckman counter.
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Results
The results for the incorporation of tritiated praline and the increase in collagen synthesis are shown in Table 1.
Table 1
Figure 5 plots the synthesis of collagen (DPM) as a function of the concentration of Pal-Gly-His-Lys. The horizontal line corresponds to the control, i.e. the synthesis of collagen in the absence of added Pal-Gly-His-Lys. The concentrations of the test substances are plotted on a logarithmic scale.
Figure 5: Synthesis of collagen (incorporation of tritiated praline)
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Conclusion
As Figure 5 shows, the addition of the peptide Pal-Gly-His-Lys to the culture medium stimulated the synthesis of collagen. The dose/response curve follows the usual form, with the optimum concentration located at about 300 µg/l of peptide, when the stimulation was about 350% of baseline.
* In vitro stimulation of the synthesis of GLYCOSAMINOGLYCANS
[Study carried out by BioAlternatives]
* Principle
Human dermal fibroblasts were cultured in an optimum growth medium (10% SVF) and then exposed to rising concentrations of the Pal-Gly-His-Lys peptide. After incubating for 48 hours, D-(6-³H) glucosamine (radiolabeled with tritium) was added. Twenty-four hours later, cells were extracted in order to isolate the macromolecules. The radioactivity count was used to determine the synthesis of GAG.
* Results
After addition of the peptide to the culture medium, there was no sign of cytotxicity and no change in the morphology of the cells. In contrast, there was obvious stimulation of the synthesis of fresh molecules of GAG, one third of which took the form of hyaluronic acid.
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Figure 6 shows the increase in the incorporation of radioactivity by GAG molecules at different concentrations of Pal-Gly-His-Lys. Increases of up to 146% above baseline (untreated control) were observed (p<0.001) at a concentration of 5 ppm of peptide, which was equivalent to 5% BIOPEPTIDE-CL.
* Conclusion
The active peptide of BIOPEPTIDE-CL, Pal-Gly-His-Lys not only stimulated the synthesis of collagen in the fibroblasts; it also induced the synthesis of the other macromolecules in the connective tissue of the dermis, the glycosaminoglycans (GAG), including hyaluronic acid. This means that it can participate in restoring the three-dimensional, functional structure of the dermis, as well as restoring its elasticity and firmness, its degree of hydration, thickness and, consequently, its ability to repair actinic damage (wrinkles, roughness) caused by age, sun and the environment. This will be discussed in the sections which follow.
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3.2 Ex vivo Histology examinations of biopsies
· * Effect of a cream containing 5% BIOPEPTIDE-CL compared to a cream
containing 500 ppm of retinoic acid (vitamin A acid versus a placebo.
We carried out a study using histological methods to investigate the behavior and activity of the peptide Pal-Gly-His-Lys on the skin after topical application. We chose to compare this activity to that of retinoic acid (a documented positive control), in the hope of finding that BIOPEPTIDE-CL and retinoic acid (the use of which is prohibited for cosmetic purposes) had similar activities.
* Principle of the study
Selected sites on the skin were exposed to repeated, controlled UVA radiation in order to produce a harmful effect on the collagen in the dermis. The histological staining of the biopsy samples was used to measure the amount of collagen present in the dermis. Macrophotography was also used to compare the thickness of the epidermis quantitatively at different stages of the study.
The carious cases studied were:
· * Control skin, before exposure to UVA
· * Skin exposed to UVA, untreated
· * Skin exposed to UVA, placebo treated
· * Skin exposed ti UVA, treated witht the cream containing 5% BIOPEPTIDE-CL?
? In the form marketed, BIOPEPTIDE-CL contains 100ppm Palmitoyl-Gly-His-Lys. The 5% Biopeptide-CL used in this study contains 0.0005% Palmitoyl-Gly-His-Lys, which is 100 times lower than the concentrationof the retinoic acid used.
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* Protocol
The skin areas wer exposed to UVA (2.7 Joules/cm²5min), for one week and treated after each exposure with the various substances listed.
At the end of the first week, biopsies samples were taken for histological and macrophotographic examination.
The treatments, with the various substances, were continued for a second week without further exposure to UVA. More biopsy samples were then taken.
This made it possible to interpret the findings in terms of a “protective effect” at the end of the first week and a “repair effect” at the end of the second week.
* Results
After one week of exposure plus treatment
The two plates which follow show the destructive effects on collagen of exposure to UVA: the blue staining, which is indicative of the amount of collagen present, is almost completely absence on plate 2 (skin exposed without treatment). After scoring by an expert (degree of staining on several biopsies and a large number of photographs), a 41 5 degradation of the collagen after exposure to UVA was detected.
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Application of the placebo to the zones exposed to the UVA for one week gave only slight protection to the collagen in the dermis: 31% degradation.
In contrast, BIOPEPTIDE-CL treatment preserved the collagen virtually intact: only 14% degraded (versus 41% if untreated), Plate 3.
Retinoic acid also protected the collagen, only 1% degraded, Plate 4.
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Measuring the thickness of the epidermis provides an initial explanation for these figures: whereas there was virtually no difference in the thickness of the epidermis at the untreated and placebo-treated sites, the areas treated with the cream containing BIOPEPTIDE-CL or with the retinoic acid, displayed thickening of epidermis by about 70 and 120% respectively. This thickening of the epidermis enhances its protective potential.
* Effects observed after two weeks’ of treatment
After the end of the exposure to UVA (repeated exposure for 1 week), the skin regenerated itself and repaired the damaged sustained. The following findings show that the repair process, particularly the reconstitution of collagen, was accelerated by treatment with either BIOPEPTIDE-CL or retinoic acid.
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The quantity of collagen found by the histological methods was the same for the exposed sites which were
Either untreated or treated with the placebo, but was 10% higher after treatment for one week with the active substances Biopeptide-CL or retinoic acid. Similarly, the thickness of the epidermis increased at the same rate with both Biopeptide-CL and retinoic acid, whereas the remnant effect of the placebo on this parameter was less marked (Table 2).
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Conclusion
The protocol and method of this study has confirmed the findings reported by Kligman and the other authors cited:
· * exposure to UV damages the dermal tissue
· * retinoic acid was capable of stimulating the in vivo synthesis of collagen and enhancing its repair effect on the skin
The activity of BIOPEPTIDE-CL is exactly the same in all respects as that of retinoic acid, the use of which is prohibited for cosmetic purposes. Furthermore, it has activity that is comparable in quantitative terms on the restoration of the epidermis, which is linked to the synthesis of collagen.
The Histological findings confirm the biological activity of this natural synthetically produced peptide, for skin care. Toxicological studies of BIOPEPTIDE-CL also show that, in contrast to retinoic acid, it has no adverse effects: no irritant or sensitizing effects or mutagenicity. In contrast to retinoic acid, BIOPEPTIDE-CL does not have a dehydrating effect, indeed quite the contrary because it is dissolved in an excipient with high moisturing potential (glyceryl polymethacrylate).
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3.3 In vivo studies
In view of the in vitro and ex vivo (on biopsies) effects of BIOPEPTIDE-CL, we investigated the cosmetic effects of the peptide incorporated into skin care creams. The parameters investigated were the thickness of the skin and the anti-wrinkle effect.
Increased synthesis of sub-cutaneous collagen could contribute to firming and restructuring this tissue. It has been shown that aging skin gets thinner, and anti-aging treatment is intended to restore the structure of the skin. Vitamin A and its derivatives have been reported to be effective in thickening the dermis. (Fthenakis et al., 19991).
* Study of skin thickening by type A ultrasound scanning (DERMASCAN)
* Principle
The method is based on the principle of an ultrasound probe. Some of these ultrasounds are reflected at each interface between two structures with different acoustic impedance (air/skin surface, dermis/hypodermis junction).
The ultrasound wave is a mechanical form of energy with a characteristic frequency. The lower its frequency, the deeper it is propagated into the skin. For this study we used a very high frequency probe (20 MHz) in order to investigate the surface layers of the skin (epidermis/dermis).
* Panel
The study included 23 female subjects between 35 and 58 years of age. Each individual was given a cream containing 4% BIOPEPTIDE-CL and a placebo. For four weeks the creams were applied daily to the inner surface of the forearm – the placebo to the left arm and the test substance to the right arm (or vice versa, as randomly selected by the investigator, which was unknown to the volunteers).
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* Results
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