Visible and measurable proof of anti-wrinkle efficacy

An in vivo, clinical study was carried out with 2 groups of 23 volunteers aged 39 to 74. These women applied a cream containing the peptides in Prolastil E-50 to one-half of their face and used a placebo on the other half twice a day for 56 days. Anti-wrinkling and lifting efficacy was assessed by profilometry and photography. The results were compared to the untreated half. Thesed results showed a significant reduction the depth, volume and surface of deep wrinkles – up to a 45% reduction.

Effect on skin elasticity and tone

The same volunteers were also evaluated for the ability of the DermLastyl peptides to improve skin elasticity and tone. Before and after photographs showed a significant improvement in elasticity and tone during the 2 month treatment period.

The peptides used in Prolastil E-50 display major and significant improvements in elasticity and tone

Skin Study- Prolastil E-50 Skin Regeneration

An initial phase scientific study of Prolastil E-50 face cream was conducted by Dr. Steven Lamm to assess tolerance, sensitivity and skin compatibility. A total of 28 volunteers, 25 women and 3 men, applied Prolastil E-50 face cream twice per day for 42 days. They were assessed throughout the study for tolerance and compatibility of the formulation. With the exception of one subject experiencing a slight breakout, all subjects tolerated the product well with no observed redness or irritation.

The subjects also performed a self-assessment of the texture, enhanced elasticity, hydrating, moisturizing, glow and greasiness of the cream.

The majority of those participating in the study stated that the use of Prolastil E-50 cream resulted in significant improvement in their skin’s texture and elasticity, a hydrating/moisturizing effect that lasted throughout the day, a glow to the skin upon application, and that the non-greasy composition allowed makeup directly over the cream. (See Figure 1).

Some participants in the study received unsolicited comments: “What are you doing differently for your skin?” Subjects with fair complexions claimed the cream lightened their skin. Most concluded they were experiencing a more youthful appearance.

Dr. Lamm has determined that based on the very positive effects of this initial stage tolerability/sensitivity/compatibility study, Prolastil E-50 deserves a more rigorous scientific evaluation.

Other Studies

In order to demonstrate the effectiveness of the ingredients used in Prolastil E-50, both Laboratory (in vitro) and clinical studies with volunteers (in vivo) were carried out. These studies show that the active ingredients in Prolastil E50 reduce the appearance of wrinkles, with a reduction of deep wrinkles by 45% within 2 months. Tropoelastin, the unique and proprietary ingredient in Prolastil E-50, was shown in studies using human skin to be able to penetrate into the skin and once within the skins matrix, become incoporated to help prevent the onset of new wrinkles.

Synthesis of Matrix Macromolecules

An in vitro study to was carried out to determine the ability of the Oligopeptides used in Prolastil E-50 to stimulate fibroblasts (the skin cells that make new propteins such as collagen and elastin). This study showed that human fibroblasts incubated in the laboratory for 72 hours with Palmitoyl Oligopeptides-3 were stimulated to synthesize new collagen, fibronectin and hyaluronic acid, all essential components of the skin matrix. Collagen and hyaluronic acid production were stimulated by 40 – 50% when using a 5% peptide concentration.

Stimulation of Gene Expression

A further study was carried out to measure the stimulation of dermal and epidermal genes by the peptides present in Prolastil E-50, using a method called DNA-Array with skin cell cultures to measure stimulation. This study showed that Prolastil E-50's active peptides help the skin’s natural regeneration process.

RESULT - The profile of gene activation by the peptides in Prolastil E-50 complement the skin's natural mechanisms for regeneration

Visible and measurable proof of anti-wrinkle efficacy

Effect on skin elasticity and tone

The peptides used in Prolastil E-50 display major and significant improvements in elasticity and tone

Elastin Uptake and Incorporation

Although large molecules such as tropoelastin are not readily taken up by skin, studies carried out on human volunteers have shown that elastin can penetrate to the deepest layers of the stratum corneum; the topmost layer of skin. Table 1 shows the results of a study of the distribution of acetate, laurate, and palmitate derivatives of elastin in the stratum corneum. Both the laurate and palmitate esters of elastin are present in layers 6 –14 of the stratum corneum after 1 hour of exposure. (Usher, T. C. US Patent #4,659,740).

Table 1. Uptake of elastin derivatives by human skin

Usher, Thomas C. #4,659,740

Once the tropoelastin enters the skin, studies of skin cell cultures have revealed that the elastin is incorporated into the matrix surrounding the cells by crosslinking. The results shown in Table 2 establish that cells in culture can incorporate up to 12% of exogenously supplied tropoelastin into the extracellular matrix. Up to 4.6 ug of elastin can be incorporated into a layer of 400 cm² of cells within 24 hours of application. This corresponds to an estimated elastin replenishment rate of 1.7 mg of facial elastin per year, or 7% of the total elastin estimated to be lost in a lifetime.

Table 2. Incorporation of tropoelastin in cultured cells

*Dose is the amount of tropoelastin in micrograms applied to 400 cm²cultured cells (equivalent to the surface area of an average face) and incorporated after 24 hours. From Stone, et. al. 2001

Key research findings about Prolastil E-50™

Prolastil E-50™ can penetrate living skin within 24 hours.
When applied to the epidermis of human skin, absorption of the Prolastil E-50™molecule occurred for 24 hours following application.
When skin was stained and studied under a microscope following application, tropoelastin could be seen penetrating into the epidermis.

The above results demonstrate that Prolastil E-50, is one of the most effective anti-wrinkle and anti-aging formulas on the market today.

Prolastil E-50™ also contains the same type of bioactive peptide products found in many popular brands which provides skin cell stimulation from these peptides. Not only can SKIN™ by Nulastin™ give your skin the additional elastin it needs in the most effective form, but it also stimulates new collagen synthesis with the proprietary peptides included in the formula.

Clinical research further supports the positive effects of Prolastil E-50™.

Observing use across the board and confirming lasting results among subjects helps to determine the success-fulness of a product. Clinical trials show that the active peptides and tropoelastin in Prolastil E-50™ possess the ability to:

Be absorbed by the skin
Participate in the biological processes that help prevent wrinkles
Make an observable difference in appearance and facial rejuvenation when tested by volunteers

The results of these studies confirm the effectiveness of the active ingredients contained in Prolastil E-50™, guaranteeing that the product will help improve the condition of one’s skin when it comes to wrinkles, signs of aging, crows feet, scars and more. You will see and feel the results.

Matrix Design

TECHNICAL DESCRIPTION

BIOMATERIALS IN Wound Healing and Tissue Regeneration

Burt D. Ensley, Ph.D.

The cells in the skin and other organs are both attached to each other and supported by a structure comprised of Extracellular Matrix Proteins, primarily different types of collagen, elastin, fibronectin and laminin. Collagens are a family of fibrous proteins that are the most abundant proteins in mammals, constituting about 25 percent of their total protein. Elastin, present in elastic fibers of tissues such as blood vessels and skin, gives these tissues the ability to recoil after stretching. Elastin in its native state is an extensively cross‑linked polypeptide and approximately one third of the amino acids are glycine, 10‑13 percent are proline, and over 40 percent are other amino acids with hydrophobic side chains.

Laminin is a major component of basement membranes made by epithelial cells. Laminin is composed of three different subunits in a cross‑linked structure. Fibronectin is a cell‑surface and blood glycoprotein. It is present in an insoluble form at the cell surface and in connective tissue and in a soluble form in blood plasma.

Human Extracellular Matrix proteins can be synthesized by genetically modified organisms, and the extracted proteins crosslinked and used as biomaterials to form scaffolds or matrices for wound healing, tissue and organ regeneration. The primary challenge in the production of these specialized materials is the synthesis and purification of sufficient quantities and quality at a reasonable cost. Human collagen is commercially available and is used as a dermal filler called Cosmoderm^Ò (Inamed Aesthetics)^, but none of the other ECM proteins has a commercial source for bulk product.

Recombinant Human Collagen. The production of recombinant collagen, a complex, triple chain helical molecule with several subtypes, uses an engineered yeast strain to synthesize the complete macromolecule (J. Myllyharju et. al. 2000. Biochem Soc. Trans. 28: 354-357). The completely formed fibrils are purified from the yeast cells. The resulting material can be formed into porous matrices and used in the repair of cartilage and bone (R. Spiro, et. al. 2002 Eur. Cells Mat. 4(1): 16). Collagen has also been used as a matrix to accelerate the healing of persistent skin wounds such as venous ulcers.

Recombinant Human Tropoelastin. The human tropoelastin gene has been cloned into E. coli and r-tropoelastin produced in small quantities by fermentation (Indik, et. el. 1990. Arch. Biochem. 280: 80-86). The resulting purified material has been shown to be water soluble and to serve as a substrate for the crosslinking enzyme lysyl oxidase (D. Bedell-Hogan, et. al. 1993. J. Biol. Chem. 268: 10345-10350). Research has confirmed that recombinant human tropoelastin is incorporated into tissues by rat fibroblast cell cultures. These studies also demonstrated that certain tropoelastin isomorphs are more efficiently incorporated into the tissue matrix (H. Hsiao, et. al. 1999. Connective Tiss. Res. 40: 83-95).

Elastin Isomorphism. Proteins in the human body are encoded by large genes carried on DNA. During the process of protein synthesis the DNA is not translated into messenger RNA in a continuous process like a tape recording, but rather in a series of discrete pieces called Exons. The exons are then spliced together to form the message directing the synthesis of the protein. For example, the DNA encoding human tropoelastin is over 45,000 bases in length, but the 34 exons that make up the actual mRNA coding sequence are in total only about 2,200 bases long (Bashir, et. al. 1989. J. Biol. Chem. 264: 8887-8891). This process can also give rise to polymorphism, or different forms of the same protein. If the mRNA is not spliced together exactly the same way every time, slightly different proteins will be produced when the mRNA is translated into protein. These different forms of what is essentially the same protein are referred to as isomorphs. The process resulting in the synthesis of elastin in the body and its potential for polymorphism is shown in Figure 1.

Figure 1. The human tropoelastin gene, mRNA synthesis and alternate exon splicing to form polymorphs.

Human tropoelastin has been known to be isomorphic for 20 years (Indik, et. al. 1987. Proc. Natl. Acad. Sci. USA 84: 5680-5684), and 9 different isomorphs of elastin were identified in 1988, including different isomorphic compositions of distinct tissues. (Fazio, et. al. 1988. J. Invest. Dermatol. 21: 458-464.). The relative abundance of 8 of the tropoelastin exons were measured in human skin fibroblast, placental and fetal aortal tissues. Each tissue contained a unique tropoelastin mRNA profile.

The absence of some of these isomorphs affects elastogenesis (elastin synthesis and deposition) and can result in serious human health consequences. A common mutation in the elastin gene that causes a deletion of exons 16 and 17 is associated with supravalvular aortic stenosis, a severe thickening of the artery wall and narrowing of the artery (Urban, Z et. al. 2001. Hum. Genet. 109: 512-520). And a deletion of exon 30 in the elastin gene has been linked to abnormal elastin deposition and causes autosomal dominant cutis laxa (Kozel, B. et. al. 2003. J. Biol. Chem. 278: 18491-18498). Clearly, elastin polymorphisms have a role in the deposition, crosslinking and function of elastin in tissues, but the specific role of the individual elastin polymorphs is unknown.

In unpublished research on elastin polymorphism in human skin, Matrix Design has identified 29 unique polymorphs of elastin in 59 clones from 4 individuals; each individual’s pattern was unique as shown in Figure 2. The surprising and extensive polymorphism in the elastin mRNAs in a single tissue (skin) between different apparently healthy individuals illustrates the genetic diversity at the tropoelastin mRNA level between individuals: a large number of different forms of elastin are synthesized in human skin.

Matrix Design intends to use this knowledge base in guiding the design of the next generation of high performance skin care, wound healing and tissue regeneration ingredients based on tropoelastin. The elastin isoforms identified from ongoing studies will be synthesized and used to evaluate their performance in accelerated tissue repair and regeneration in vitro. based on the data collected via our genetic analyses. This information will be applied to the engineering of new, more complete matrix materials that can be applied to stimulate wound repair, tissue regeneration and skin care in general. This approach represents the next step in skin care taken to the organ, molecular and genetic level.

These wound healing and tissue regeneration compositions will be composed of selected elastin isoforms combined with cross-linking reagents, other ECM molecules and growth factors. These components will form a gel and be applied to and become a part of the healing wound. This gel can be specifically tailored to promote recruitment of skin cells, regeneration of injured tissue and the growth of new skin. Independent research has already shown that the strength, elasticity, crosslinking potential and other physical and biochemical behavior of elastin can be varied and possibly controlled by incorporating various polymorphic forms of elastin in a wound healing matrix (Hsiao, et al. Connect Tiss Res 40(2):83-95, 1999).

Figure 2. Exon Splice Variants of elastin in human clones from 4 individuals.

PERSONAL CARE PRODUCTS

Exposure of the skin to sun, wind, and other factors leads to skin aging - loss of moisture, elasticity, skin tone and texture as degradation of certain skin proteins takes place. The connective tissue found in skin is an intricate mesh of interacting protein molecules that constitute the extracellular matrix. Loss or alteration of these molecules contributes to the appearance of “aged” skin.

Components of the extracellular matrix of animals have been often incorporated into cosmetic compositions. In some instances, normally cross‑linked and insoluble proteins such as elastin are rendered soluble using a variety of chemical and enzymatic methods. The rationale behind these procedures is that soluble elastin derivatives will penetrate into the skin to a greater degree than cross‑linked elastin to compensate for loss of elastin during skin aging. However, the chemical and enzymatic methods used in solubilization induce extensive chemical and structural changes in the elastin molecule itself. In addition the soluble precursor of insoluble elastin, called tropoelastin, is very difficult to extract and is destroyed during the use of conventional methods for solubilizing elastin.

Another consideration affecting the use of proteins such as elastin as skin care ingredients is unwanted allergic responses in the skin. This is particularly troublesome since the elastin used in most cosmetics is derived from the neck tendons of young calves. Non-human sources of many proteins are potent allergens and cannot be used in unmodified form in humans. The use of authentic human extracellular matrix proteins in cosmetics avoids these problems. A biotechnology based process to manufacture human elastin or tropoelastin provides a unique, functional source of this matrix protein for cosmetic compositions. The material displays several properties that make it far superior to any conventional source of this material.

The ability of cells to synthesize elastin declines with age (Fazio, M. J. et. al. 1988. Laboratory Investigation. 58: #3; 270-277.) and this decline may contribute to the formation of wrinkles in the skin. Elastin synthesis in the cells results in a protein with a molecular weight of approximately 72,000 called tropoelastin composed of a structural protein and signal peptide. This protein is processed after synthesis by cleavage of the signal peptide via a signal peptidase and the formation of crosslinks between individual elastin molecules through the action of the enzyme lysyl oxidase to form a tissue matrix (Figure 3).

Figure3. Synthesis and polymerization of elastin. A protein molecule is synthesized in the cell as the precursor tropoelastin and exported to the extracellular environment. A signal peptide of 26 amino acids is cleaved by a signal peptidase from the precursor molecule in step 1. Individual elastin molecules are crosslinked at lysine residues in step 2 by the enzyme lysyl oxidase. Further crosslinking shown in step 3 results in the formation of an insoluble, elastic matrix surrounding the cells and providing strength and elasticity to the tissue.

Bovine Elastin. A current source of elastin for cosmetic preparations is prepared from bovine tissue. An example is the product Hydroelastin used in Elastin and Collagen Firming Treatment by Rachel Perry. However, the crosslinking of elastin after its synthesis in the skin makes it highly insoluble. Elastin is released from bovine tissue by exposing it to hydrolyzing conditions such as alkali and proteolytic enzymes that degrade the polypeptide backbone of the molecule without cleaving the crosslinks formed in the final processing step, leaving a mixture of crosslinked peptides. This procedure is shown in Figure 4.

Figure 4. Preparation of soluble elastin from tissue. Exposure to proteolytic enzymes and alkaline conditions is used to break up the solid tissue by cleavage of the polypeptide backbone into much smaller fragments, with some increased water solubility. The final product is a mixture of small, crosslinked peptides.

The final product has increased solubility compared to an elastin matrix, but the material produced by this method bears a much greater resemblance to beef stew than to native elastin. It is unlikely that this crosslinked peptide material is absorbed by the skin, nor is this product recognized by the skin’s biochemical machinery. This form of hydrolyzed elastin is also likely to be washed off the skin surface immediately upon cleansing.

Human Elastin. To date, human elastin has not been available as a cosmetic ingredient. Human tissue is not used as a source of hydrolyzed elastin, although placental tissues have been occasionally used in cosmetic preparations. Developments in the field of molecular biology now make it possible to produce human elastin or the tropoelastin precursor in transgenic microorganisms or plants. The significant advantages provided by this source of elastin would include:

 A truly water soluble and intact protein

 Higher likelihood of being absorbed by the skin

 Compatible with the biochemistry of the human skin

 Available to be incorporated in the tissue matrix through crosslinking

 More esthetically acceptable

 Identical to the human material

 Available as the tropoelastin precursor

 Can be produced as isomorphs

Elastin Penetration and Incorporation. Although proteins such as elastin are not readily taken up in human skin, in vivo studies carried out using labeled elastin on human volunteers have shown that simple derivatives of elastin can cause this molecule to penetrate to the deepest layers of the stratum corneum; the topmost layer of skin. Table 1 shows the results of a study of the distribution of acetate, laurate, and palmitate derivatives of elastin in the stratum corneum. Both the laurate and palmitate esters of elastin are present in layers 6 –14 of the stratum corneum after 1 hour of exposure. (Usher, T. C. US Patent #4,659,740).

Table 1. Uptake of elastin derivatives by human skin.

Elastin Derivative	Penetration into Stratum Corneum
	% Distribution in Layer
	1-2	3-5	6-8	9/11	12-14
acetate	88	9	2	1	0
laurate	53	10	21	11	4
palmitate/oleate	26		19.6		1.7

Usher, Thomas C. #4,659,740

Matrix-sponsored research performed at Thomas Jefferson University has confirmed the above results and demonstrated that synthesized human tropoelastin can be absorbed and incorporated into the epidermis of human skin. Live human skin derived from dermal surgery was maintained in a growth chamber and a 0.25 cm²area was exposed to 10 mg of tropoelastin for 2 to 24 hours, washed 6 times with PBS, and tropoelastin stained with a monoclonal antibody against tropoelastin. The results are shown in Figure 5.

Figure 5. Uptake of tropoelastin over 24 hours followed by staining with antibodies against tropoelastin.

Once the tropoelastin enters the skin, in vitro studies using cell cultures have revealed that the elastin is incorporated into the matrix surrounding the cells by crosslinking. The results shown in Table 2 establish that fibroblast cells in culture can incorporate up to 12% of exogenously supplied tropoelastin into the extracellular matrix. Up to 4.6 ug of elastin can be incorporated into a layer of 400 cm² of cells within 24 hours of application. This extrapolates to an estimated elastin replenishment rate of 1.7 mg of facial elastin per year, significantly exceeding the natural depletion rate.

Table 2. Incorporation of tropoelastin in cultured cells

Tropoelastin Dose*	Amount (Percent) of Tropoelastin Incorporated Into Cells
8 ug	.96 ug (12%)
31 ug	1.86 ug (6%)
77 ug	4.62 ug (6%)

*Dose is the amount of tropoelastin in micrograms applied to 400 cm² cultured cells (equivalent to the surface area of an average face) and incorporated after 24 hours. From Stone, et. al. 2001.

The proprietary data we have collected indicates that the tropoelastin isomorphic composition of an individual is unique; similar to fingerprints or iris pigmentation. Any population group or even individual could be chosen to serve as a model for a tropoelastin cosmetic composition, their mRNA isolated from skin cells, and the precise isomorphic composition of the tropoelastin mRNA determined. A series of tropoelastin genes would then be synthesized based on the genetic information obtained and a tropoelastin composition modeled after that of the chosen population group or individual produced. A schematic of this process is shown in Figure 6.

Figure 6. Production of polymorphic tropoelastin. A human population group is sampled and the mRNA isolated in step 1. The RNA is amplified in step 2. The relative abundance of each type of elastin mRNA is deduced in step 3. A gene encoding each elastin isomorph is custom synthesized in step 4, and introduced into an expression host . The discrete elastin molecules are produced by the transgenic host in step 5, and the proteins formulated to match the original relative mRNA abundance of the population sample. The formulated protein is incorporated into a personal care product and bottled in step 6.