Ghk Cu Synthesis Essay

Skin Biology, Research & Development Department, 4122 Factoria Boulevard SE, Suite No. 200, Bellevue, WA 98006, USA

Copyright © 2014 Loren Pickart et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

During human aging there is an increase in the activity of inflammatory, cancer promoting, and tissue destructive genes plus a decrease in the activity of regenerative and reparative genes. The human blood tripeptide GHK possesses many positive effects but declines with age. It improves wound healing and tissue regeneration (skin, hair follicles, stomach and intestinal linings, and boney tissue), increases collagen and glycosaminoglycans, stimulates synthesis of decorin, increases angiogenesis, and nerve outgrowth, possesses antioxidant and anti-inflammatory effects, and increases cellular stemness and the secretion of trophic factors by mesenchymal stem cells. Recently, GHK has been found to reset genes of diseased cells from patients with cancer or COPD to a more healthy state. Cancer cells reset their programmed cell death system while COPD patients’ cells shut down tissue destructive genes and stimulate repair and remodeling activities. In this paper, we discuss GHK’s effect on genes that suppress fibrinogen synthesis, the insulin/insulin-like system, and cancer growth plus activation of genes that increase the ubiquitin-proteasome system, DNA repair, antioxidant systems, and healing by the TGF beta superfamily. A variety of methods and dosages to effectively use GHK to reset genes to a healthier state are also discussed.

1. Introduction

According to the Administration on Aging (http://www.aoa.gov/), there were 39 million people aged 65 and older in 2009 which constituted 12% of the American population. By 2030 it is expected that 19% of the population will be over 65. With life expectancy continuing to increase, we may expect that this trend is here to stay. Unfortunately, with advanced age comes not only wisdom but also many age-related pathological conditions that account for the high rates of hospitalization, increased cost of health care and decreased quality of life. Today, more than ever, there is an urgent need to find safe, easy-to-administer, cost-effective methods, which could not only delay the onset of the age related diseases, but also restore health.

It now becomes increasingly clear that the primary cause of human aging and its attendant diseases is changes in the activity of the human genome. During aging there is an increase in the activity of inflammatory, cancer promoting, and tissue destructive genes plus a decrease in the activity of regenerative and reparative genes [1].

The most exciting discovery of the past decades is that these changes in gene activity can be reversed, often by quite simple and natural molecules [2]. Recent discoveries on the actions of the human tripeptide GHK (glycyl-L-histidyl-L-lysine) to reset gene expression of human cells to a more healthy state may open a door to the therapeutic resetting of genes in the elderly. This can be useful as a preventative measure and a complimentary treatment for conditions typically associated with aging such as cancer, Alzheimer’s, chronic obstructive lung disease (COPD), nephropathy, and retinopathy.

GHK was discovered during studies comparing the effect of human plasma from young persons (age 20–25) to plasma from older persons (age 50–70) on the functioning of incubated slices of human hepatic tissue. The younger plasma more effectively induced a profile associated with youth by suppressing fibrinogen synthesis. The active factor was found to be GHK. Since then numerous studies over the course of four decades demonstrated that this simple molecule improves wound healing and tissue regeneration (skin, hair follicles, bones, stomach, intestinal linings, and liver), increases collagen and glycosaminoglycans, stimulates synthesis of decorin, increases angiogenesis, and nerve outgrowth; possesses antioxidant and anti-inflammatory effects, and increases cellular stemness and the secretion of trophic factors by mesenchymal stem cells [3–6].

GHK’s actions on gene expression were determined by the Broad Institute and, using their data, we determined that GHK increased or decreased gene expression (UP or DOWN more than 50%) in 32.1% of the human genes [7]. In a recent gene study, the Broad Institute’s Connectivity Map was used to find potential therapeutic agents for aggressive, metastatic colon cancer [8]. The gene analysis computer program selected GHK from 1,309 bioactive molecules as the best choice to reset the diseased gene patterns to a healthier pattern. When three lines of human cancer cells (SH-SY5Y neuroblastoma cells, U937 histolytic cells, breast cancer cells) were incubated in culture with 1 to 10 nanomolar GHK, the programmed cell death system (apoptosis) was reactivated and cell growth inhibited [9]. When cells derived from the damaged areas of the lungs of COPD patients were incubated with 10 nanomolar GHK, the tripeptide recapitulated TGF beta induced genes expression patterns which led to the organization of the actin cytoskeleton and elevated the expression of integrin. This restored proper collagen contraction and remodeling by lung fibroblasts [10]. These results, combined with GHK’s broad spectrum of positive actions on many systems that maintain human health, suggest that therapies using GHK might provide health benefits to the elderly.

In this paper, we discuss the following actions of GHK on genes important in healthy aging.

(1) The Suppression of Fibrinogen Synthesis. Fibrinogen is an excellent predictor of mortality especially in patients with cardiovascular complications [11, 12]. GHK was isolated as a plasma factor that suppressed fibrinogen synthesis in liver tissue and in mice.

(2) Activation of the Ubiquitin/Proteasome System (UPS). The UPS removes damaged proteins. Higher activities of the UPS appear to retard aging effects [13, 14].

(3) Activation of DNA Repair Genes. DNA damage is promptly repaired in young and healthy cells, however, as we age, DNA damage starts accumulating. Resetting activity of DNA repair genes can diminish deleterious effects of aging.

(4) Antioxidant Genes. Free radicals and toxic end products of lipid peroxidation are linked to atherosclerosis, cancer, cataracts, diabetes, nephropathy, Alzheimer’s disease and other severe pathological conditions of aging.

(5) Suppression of Insulin and Insulin-Like Genes. The insulin family has been proposed as a negative controller of longevity; higher levels of insulin and insulin-like proteins reduce the lifespan [15].

(6) Tissue Repair by TGF Superfamily. General tissue repair by the TGF superfamily as exemplified by COPD (chronic obstructive pulmonary disease).

(7) Cancer Controlling Genes. Caspase, growth regulatory, and DNA repair genes are important in cancer suppression.

In addition to discussing GHK actions in this paper, we suggest administrative methods and dosages that should be effective in humans.

2. Methods and Results

2.1. Gene Expression Analysis

The connectivity map was used to acquire our gene expression data (retrieved March 5, 2013) [16]. Within the Connectivity Map repository there are three GHK gene signatures. Each signature was produced using the GeneChip HT Human Genome U133A Array. GHK was tested on 2 of the 5 cell lines used by the Connectivity Map. Two of the profiles were created using the PC3 cell line while the third used the MCF7 cell line. Our studies utilized all three gene expression profiles.

This genomic data was then analyzed using GenePattern [17]. The CEL files were processed with MAS5 and background correction. Files were then uploaded to the ComparativeMarkerSelectionViewer module in order to view fold changes for each probe set.

Since many probe sets map to the same gene we converted the fold changes in m-RNA production produced by GenePattern to percentages and then averaged all probe sets representing the same gene. It was determined that the 22,277 probe sets in the Broad data represent 13,424 genes. This ratio (1.66) was used to calculate the overall number of genes that are affected by GHK at various cutoff points (rather than probe sets). The number of genes stimulated or suppressed by GHK with a change greater than or equal to 50% is 31.2%.

2.2. Fibrinogen Suppression

Fibrinogen consists of three polypeptide chains; alpha, beta, and gamma. GHK strongly suppresses the gene for the beta chain of fibrinogen. A lack of adequate FGB will effectively stop fibrinogen syntheses since equal amounts of all three polypeptide chains are needed to produce fibrinogen. See Table 1.

Table 1: GHK and fibrinogen.

GHK also suppresses the production of the inflammatory cytokine interleukin-6 (IL-6) which is a main positive regulator of fibrinogen synthesis, through its interaction with fibrinogen genes [18]. In cell culture systems, GHK suppresses IL-6 secretion in skin fibroblasts and IL-6 gene expression in SZ95 sebocytes [19, 20].

In summary, the effects of GHK on the FGB gene plus its effects on IL-6 production imply a suppression of overall fibrinogen production.

2.3. Ubiquitin/Proteasome System

GHK stimulated gene expression in 41 UPS genes while suppressing only 1 UPS gene. See Table 2.

Table 2: Ubiquitin/proteasome system and GHK.

2.4. DNA Repair Genes

GHK was primarily stimulatory for DNA repair genes (47 UP, 5 DOWN). See Tables 3 and 4.

Table 3: GHK and DNA repair.

Table 4: The most affected DNA repair genes.

2.5. Antioxidant Genes

Among the 13,424 available genes in the Broad Institute data, we were able to identify 14 antioxidant genes in which GHK stimulates as well as two prooxidant genes that GHK suppresses. GHK increases the expression of the oxidative/inflammatory gene NF-κB2 103% but also increases the expression of two inhibitors of NF-κB, TLE1 by 762% and IL18BP by 295%, thus possibly inhibiting the activity of the NF-κB protein. See Table 5.

Table 5: GHK effects on antioxidant genes.

2.6. Insulin and Insulin-Like System

GHK stimulates 3 genes in this system and suppresses 6 genes. See Table 6.

Table 6: GHK and insulin/insulin-like genes.

3. Discussion

Even though numerous and diverse beneficial effects of GHK have been known for decades, it was not clear how one simple molecule could accomplish so much. The use of gene expression data greatly extends our understanding of GHK’s effects and its potential treatments of some of the diseases and biochemical changes associated with aging. As a potential therapeutic agent GHK has a clear advantage over many other active chemicals that may also show promising results in gene profiling experiments, its gene modulating effects correspond to findings from in vivo experiments. When GHK is administered internally to an animal, it induces actions throughout the body.

The treatment of rats, mice, and pigs with GHK was shown to effectively activate systemic healing throughout the animal. For example, if GHK is injected into the thigh muscles of rats, it induces accelerated healing in implanted Hunt-Schilling wound chambers. If the GHK is injected into the thigh muscles of mice, it accelerates the healing of an experimental full thickness surgical defect wound model on its back. If injected into thigh muscles of pigs, it induces accelerates healing of full thickness surgical defect wounds on its back [37]. If GHK is injected intraperitoneally into rats, it heals tubular bone fractures [38]. Wound healing requires activation of gene expression for numerous pathways and wound healing data confirms that GHK is able to activate gene expression in animals [39–45].

There is still not enough information to translate gene profiling data into biological effects. However, based on the documented activity of GHK in vivo, we can predict the following beneficial actions from our gene profiling data.

3.1. Fibrinogen

Fibrinogen, the protein which is used to make blood clots, is also a strong predictor of mortality in cardiovascular patients. After vascular incidents, such as myocardial infarction, fibrinogen concentrations increase sharply. The free, unclotted fibrinogen protein increases the “stickiness” of red blood cells which stack together forming rouleaux. This increases the time of the “solid” blood state which decreases blood flow through the microcirculation where blood flows like a thixotropic fluid, switching between a solid phase and a liquid phase, somewhat like toothpaste. As a solid, it stops oxygen and nutrient flow to the tissues. This, in itself, can cause tissue damage.

The gene data on GHK’s suppression of FGB (the fibrinogen beta chain) combined with its actions on lowering IL-6 secretion on fibroblasts and sebocytes appears to be sufficient to explain its lowering effect on fibrinogen.

3.2. Ubiquitin Proteasome System

The ubiquitin proteasome system (UPS) functions in the removal of damaged or misfolded proteins. Aging is a natural process that is characterized by a progressive accumulation of unfolded, misfolded, or aggregated proteins. In particular, the proteasome is responsible for the removal of normal as well as damaged or misfolded proteins. Recent work has demonstrated that proteasome activation by either genetic means or use of compounds retards aging [13, 14].

In our screening of UPS genes with a percent change of at least ±50%, GHK increased gene expression in 41 UPS genes while suppressing 1 UPS gene. Thus, it should have a positive effect on this system [13, 14, 46].

3.3. DNA Repair

It is estimated that normal metabolic activities and environmental factors such as UV light and radiation can cause DNA damage, resulting in somewhere between 1000 and as many as 1 million individual molecular lesions per cell per day. Lack of sufficient DNA repair is considered a cause of cell senescence, programmed cell death, and unregulated cell division, which can lead to the formation of a tumor that is cancerous [47–50].

GHK was stimulatory for DNA repair genes (47 stimulated, 5 suppressed) suggesting an increased DNA repair activity.

3.4. Antioxidant Defense

Free radicals and toxic end products of lipid peroxidation are linked to atherosclerosis, cancer, cataracts, diabetes, nephropathy, Alzheimer’s disease, and other severe pathological conditions of aging. Reactive oxygen species (ROS) and reactive carbonyl species (RCS) are produced in cells in small quantities under physiological conditions and play an important role in cell signaling and immune defense. A robust antioxidant network maintains balance between free radical production and scavenging, ensuring that the overall damage from free radicals is low. However, in the course of aging and in pathological conditions such as inflammation, the balance may shift toward free radical accumulation that can lead to oxidative stress and eventually to cell death [51].

GHK increases gene expression of 14 antioxidant genes and suppresses the expression of 2 prooxidant genes. It increases the expression of the oxidative/inflammatory gene NF-κB2 103% but also increases the expression of two inhibitors of NF-κB, TLE1 by 762% and IL18BP by 295%; thus, it possibly inhibits the activity of the NF-κB protein.

GHK also possesses antioxidant activities in cell culture and in vivo.

In dermal wound healing in rats, GHK, attached to biotin to bind it to collagen pads covering wounds, produced a higher production of protein antioxidants in the wound tissue. Superoxide dismutase was increased 80% while catalase was increased 56% [52, 53]. GHK reduced gastric mucosal damage by 75% against lipid peroxidation by oxygen-derived free radicals induced by acute intragastric administration of ethanol [54].

Interleukin 1 beta can induce serious oxidative damage to cultured cells [55, 56]. GHK markedly reduced oxidative damage by interleukin 1-beta to cultured insulin secreting pancreatic cells [57].

In another study, GHK entirely blocked the extent of in vitro Cu(2+)-dependent oxidation of low density lipoproteins (LDL). Treatment of LDL with 5 microM Cu(2+) for 18 hours in phosphate buffered saline (PBS) resulted in extensive oxidation as determined by the content of thiobarbituric acid reactive substances. Oxidation was entirely blocked by GHK. In comparison, copper, zinc-superoxide dismutase provided only 20% protection [58].

Acrolein, a well-known carbonyl toxin, is produced by lipid peroxidation of polyunsaturated fatty acids. GHK directly blocks the formation of 4-hydroxynonenal and acrolein toxins created by carbonyl radicals that cause fatty acid decomposition [59, 60]. GHK also blocks lethal ultraviolet radiation damage to cultured skin keratinocytes by binding and inactivating reactive carbonyl species such as 4-hydroxynoneal, acrolein, malondialdehye, and glyoxal [61].

Iron has a direct role in the initiation of lipid peroxidation. An Fe(2+)/Fe(3+) complex can serve as an initiator of lipid oxidation. The major storage site for iron in serum and tissue is ferritin and the superoxide anion can promote the mobilization of iron from ferritin which can catalyze lipid peroxidation. GHK : Cu(2+) produced an 87% inhibition of iron release from ferritin by apparently blocking iron’s exit channels from the protein [62].

3.5. Insulin and Insulin-Like Pathways

The insulin/IGF-1-like receptor pathway is a contributor to the biological aging process in many organisms. The gene expression data suggests that GHK suppresses this system as 6 of 9 of the affected insulin/IGF-1 genes are suppressed.

Insulin/IGF-1-like signaling is conserved from worms to humans. In vitro experiments show that mutations that reduce insulin/IGF-1 signaling have been shown to decelerate the degenerative aging process and extend lifespan in many organisms, including mice and possibly humans. Reduced IGF-1 signaling is also thought to contribute to the “antiaging” effects of calorie restriction [63].

3.6. COPD

COPD (chronic obstructive lung disease) is a leading cause of death in the world. It is a deadly and painful disease of the lungs that causes difficulty in breathing. In people with COPD, the tissues necessary to support the physical shape and function of the lungs are destroyed. COPD is most often caused by tobacco smoking and long-term exposure to air pollution but is also a component of normal aging. As the lungs get older, the elastic properties decrease, and the tensions that develop can result in areas of emphysema.

The most explored of GHK’s actions is the repair of damaged tissues (skin, hair follicles, stomach and intestinal linings, and boney tissue) either by the use of copper-peptide containing creams or by induction of systemic healing. Campbell et al. found that GHK’s resetting of gene expression of fibroblasts from COPD patients fits into this category of tissue repair via the TGF beta superfamily. Campbell et al. found that GHK directly increases TGF beta and other family members which activate the repair process [10].

Treatment of human fibroblasts with GHK recapitulated TGF beta-induced gene expression patterns, led to the organization of the actin cytoskeleton and elevated the expression of integrin beta1. Furthermore, addition of GHK or TGF beta restored collagen I contraction and remodeling by fibroblasts derived from COPD lungs compared to fibroblasts from former smokers without COPD.

On another note, persons with severe COPD use air inhalation systems that pump misty, water-filled air in and out of the lungs. Often steroids are added to the solution to suppress the lung inflammation, while this provides short-term help, it also inhibits lung repair. In theory, GHK could be infused into the blood stream of patients to repair the lung tissue, added to a misting solution or used in combination of a carrier like DMSO along with GHK (use a 1 : 1 molar ratio of GHK to DMSO). DMSO and GHK or GHK : Cu(2+) has always worked well together on wound healing. DMSO has been used in the past as a treatment for COPD, so there should be few safety issues.

Also, it may be possible to induce more extensive rebuilding of lung tissue. The mixture of GHK, transferrin, and somatostatin was sufficient to promote branching in the absence of serum in organ culture, all of which could be added to the misting solution [64].

3.7. Cancer

In 2010, Hong et al. identified 54 genes associated with aggressive, metastatic, human colon cancer [8]. The Broad Institute’s Connectivity Map was used to find compounds that reverse the differential expressions of these genes. The results indicated that two wound healing and skin remodeling molecules, GHK at 1 micromolar and securinine at 18 micromolar, could significantly reverse the differential expression of these genes and suggested that they may have a therapeutic effect on the metastasis-prone patients.

Normal healthy cells have checkpoint systems to self-destruct if they are synthesizing DNA incorrectly through programmed cell death or the apoptosis system. Matalka et al. demonstrated that GHK, at 1 to 10 nanomolar, reactivated the apoptosis system, as measured by caspases 3 and 7, and inhibited the growth of human SH-SY5Y neuroblastoma cells, human U937 histiocytic lymphoma cells, and human breast cancer cells [9]. In contrast, the GHK accelerated the growth of healthy human NIH-3T3 fibroblasts.

Our analysis of GHK’s actions found that it increased gene expression in 6 of the 12 human caspase genes that activate apoptosis. In 31 other genes, GHK altered the pattern of gene expression in a manner that would be expected to inhibit cancer growth. In DNA repair genes there was an increase (47 UP, 5 DOWN) [7]. These results support the idea that GHK may help slow or suppress cancer growth.

Linus Pauling’s group once used a copper tripeptide, Gly-Gly-His : Cu(2+) and ascorbic acid as a cancer treatment method. In a recent paper, we used their basic method but with GHK : Cu(2+) and ascorbic acid, which strongly suppressed sarcoma 180 in mice without any evident distress to the animals [7]. GHK altered gene expression in 84 genes (caspases, cytokines, and DNA repair genes) in a manner that would be expected to suppress cell growth. On skin, GHK seems to act most strongly in the late stage of healing, called remodeling, where cellular migration into the wound area is stopped and cellular debris is removed. The anticancer actions of small copper peptides may be a side effect of this system.

The use of GHK : Cu(2+) and ascorbic acid should be investigated in more detail. The mice treated in this manner appeared to remain very healthy and active, in contrast to the toxicities of current cancer chemotherapy.

3.8. GHK as a Clinical Treatment

GHK, abundantly available at low cost in bulk quantities, is a potential treatment for a variety of disease conditions associated with aging. The molecule is very safe and no issues have ever arisen during its use as a skin cosmetic or in human wound healing studies.

GHK has a very high affinity for Cu(2+) (pK of association = 16.4) and can easily obtain copper from the blood’s albumin bound Cu(2+) (pK of association = 16.2) [3]. Most of our key experiments used a 1 : 1 mixture of copper-free GHK and GHK : Cu(2+). In wound healing experiments, the addition of copper strongly enhanced healing. However, others often obtain effective results without added copper.

Cells within tissues are under the influence of many other regulatory molecules. Thus, GHK would be expected to influence the cells’ gene expression to be more similar to that of a person of age 20–25, an age when the afflictions of aging are very rare. Based on our studies, in which GHK was injected intraperitoneally once daily to induce systemic wound healing throughout the body, we estimate about 100–200 mgs of GHK will produce therapeutic actions in humans. But even this may overestimate the necessary effective dosage of the molecule. Most cultured cells respond maximally to GHK at 1 nanoM. GHK has a half-life of about 0.5 to 1 hour in plasma and two subsequent tissue repair studies in rats found that injecting GHK intraperitoneally 10 times daily lowered the necessary dosage by approximately 100-fold in contrast to our earlier studies [38, 65].

The most likely effective dosage of GHK was given to rats for healing bone fractures. This mixture of small molecules included Gly-His-Lys (0.5 μg/kg), dalargin (1.2 μg/kg) (an opioid-like synthetic drug), and the biological peptide thymogen (0.5 μg/kg) (L-glutamyl-L-tryptophan) to heal bones. The total peptide dosage is about 2.2 μg/kg or, if scaled for the human body, about 140 μg per injection with 10 treatments per day [38, 65].

The use of portable continuous infusion pumps for a treatment might maintain an effective level in the plasma and extracellular fluid with the need for much less GHK. Possibly the peptide could be administered with a transdermal patch [66]. Another approach could be to use peptide-loaded liposomes as an oral delivery system for uptake into the intestinal wall without significant breakdown [67, 68].

4. Conclusion

Most current theories and therapies to treat disease tend to target only one biochemical reaction or pathway. But for human aging, our data suggests that we must think of simultaneously resetting hundreds to thousands of genes to protect at-risk tissues and organs. GHK may be a step towards this gene resetting goal.

Conflict of Interests

The authors declare that there is no conflict of interests regarding the publication of this paper.

Acknowledgments

The authors would like to thank Idelle Musiek, MFA, and Genevieve Pickart, MA, for their invaluable work in the paper preparation.

Effects of GHK-Cu on MMP and TIMP Expression, Collagen and Elastin Production, and Facial Wrinkle Parameters

Travis Badenhorst1, Darren Svirskis1, Mervyn Merrilees2, Liane Bolke3 and Zimei Wu1*

1School of Pharmacy, Faculty of Medical and Health Sciences, New Zealand

2School of Medical Sciences, Faculty of Medical and Health Sciences, New Zealand

3Dermatest GmbH, Münster, Germany

Corresponding Author:
Zimei Wu
Senior Lecturer in Pharmaceutics, School of Pharmacy
University of Auckland, Private Bag 92019, Auckland 1142, New Zealand
Tel: 6499231709
Fax: 64 93677192
E-mail:[email protected]

Received Date: November 16, 2016; Accepted Date: December 20, 2016; Published Date: December 22, 2016

Citation: Badenhorst T, Svirskis D, Merrilees M, Bolke L, Wu Z (2016) Effects of GHK-Cu on MMP and TIMP Expression, Collagen and Elastin Production, and Facial Wrinkle Parameters. J Aging Sci 4:166. doi: 10.4172/2329-8847.1000166

Copyright: © 2016 Badenhorst T, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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Abstract

Background: Glycyl-L-histidyl-L-lysine-copper (GHK-Cu) is an endogenous tripeptide-copper complex involved in collagen synthesis and is used topically as a skin anti-aging and wound healing agent. However, its biological effects are yet to be fully elucidated.

Objectives: To investigate the effects of GHK-Cu on gene expression of metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs), and on production of collagen and elastin by human adult dermal fibroblasts (HDFa); and to investigate the effectiveness of topical application of GHK-Cu on wrinkle parameters in volunteers.

Methods: Cultured HDFa were incubated with GHK-Cu at 0.01, 1 and 100 nM in cell culture medium. Gene expression (mRNA) for MMP1, MMP2, TIMP1 and TIMP2 in treated and control HDFa was measured by RT-PCR. Cellular production of collagen and elastin was measured colourmetrically using commercial assay kits. Correlations between gene expression and collagen and elastin production were determined. A randomised, double-blind clinical trial involving twice daily application of GHK-Cu, encapsulated in lipid-based nano-carrier, to facial skin of female subjects (n= 40, aged 40 to 65) was run over 8 weeks. The formulation vehicle (a serum) and a commercial cosmetic product containing Matrixyl® 3000, a lipophilic GHK derivative, were used as controls.

Results: GHK-Cu significantly increased gene expression of MMP1 and MMP2 at the lowest concentration whilst simultaneously increasing the expression of TIMP1 at all the tested concentrations. All examined concentrations of GHK-Cu increased both collagen and elastin production. An increase of the mRNA expression ratio of TIMPs to MMPs was associated with an increase in collagen/elastin production. Application of GHK-Cu in nano-carriers to facial skin of volunteers significantly reduced wrinkle volume (31.6%; p=0.004) compared to Matrixyl® 3000, and significantly reduced wrinkle volume (55.8%; p<0.001) and wrinkle depth (32.8%; p=0.012) compared to control serum.

Conclusions: GHK-Cu significantly increased collagen and elastin production by HDFa cells depending on the relative mRNA expression of their TIMP(s) over MMP. Topical application of GHK-Cu with the aid of nano-carriers reduced wrinkle volume to a significantly greater extent than the vehicle alone or a commercial product containing Matrixyl 3000®, a GHK lipophilic derivative.

Keywords

GHK-tripeptide; Metalloproteinases (MMP); Tissue inhibitors of metalloproteinases (TIMP); Cell culture; Delivery; Skin barrier

Introduction

The peptide glycyl-L-histidyl-L-lysine-copper (GHK-Cu) is gaining interest as an anti-aging and wound healing bioactive agent [1-3]. GHK is capable of up and downregulating over 4000 genes [4]. Previously, the effects of GHK-Cu on collagen production and metalloproteinase (MMP) expression have been investigated in cultured rat fibroblasts and in rat wound healing models [5-7]. GHKCu functions as an activator of tissue remodelling and increases secretion of MMP2 and a number of tissue inhibitors of metalloproteinases (TIMP1 and TIMP2) in cultured fibroblasts [7,8]. MMP1, 8 and 13 degrade mainly fibrillar collagens whilst gelatinases MMP2 and 9, act on type IV collagen in the basement membrane and elastin. Increased expression of MMPs typically occurs in heightened inflammatory responses that are usually marked by opposing inhibitory processes [9]. TIMPs tightly control MMP activities through competitive irreversible inhibition, thereby controlling the breakdown and re-synthesis of the extracellular matrix [10,11].

Therefore, increased TIMP expression in the skin may have antiwrinkle benefits. While this has yet to be determined, topical application of GHK-Cu has resulted in beneficial effects on wrinkles [12]. A 12 week trial of topically administered GHK-Cu in 71 volunteers demonstrated improvements in fine lines, viscoelastic properties, thickness and density of the skin, without irritation [12]. Other trials report significant improvements in skin appearance [13], increased dermal keratinocyte proliferation and increased pro-collagen synthesis [14]. Maquart et al. found dose related effects of GHK-Cu, including increases in dry weight, total protein, collagen and glycosaminoglycan content in rat skin [15].

Topical application of GHK-Cu, amongst numerous other peptides, is widely promoted in the cosmetic industry [16]. Our previous preformulation studies, however, showed the logD of GHK-Cu at pH 4.5 and 7.4 to be -2.49 ± 0.33 and -2.49 ± 0.35, respectively, suggesting the tripeptide is highly hydrophilic [17]. Therefore, while this peptide may have considerable biological potential, the efficient trans-epidermal delivery of GHK-Cu is challenging based on its physicochemical properties. Ideally, compounds should have a moderate oil-water partition coefficient (log P) of between 1-3 and few polar centres in order to permeate into the skin [18]. To overcome the epidermal barrier, in this present study GHK-Cu was formulated into a lipophilic nano-carrier that improves delivery into the skin. Increased lipophilicity may also be achieved by combining GHK with a lipophilic moiety. For example, Matrixyl 3000®, containing a chemical combination of GHK and palmitic acid, is currently used as an active component in commercial cosmetic products.

There are potential differences associated with GHK-Cu application to rat and human fibroblasts [19]. This study therefore aimed to determine the effect of GHK-Cu, for the first time, on human adult dermal fibroblasts (HDFa), using collagen, elastin, and mRNA expression of MMP1, MMP2, TIMP1 and TIMP2 as biological markers. Further, the effect of GHK-Cu on wrinkle parameters was evaluated in volunteers, comparing a product containing Matrixyl 3000®, and a serum vehicle control, in a randomised double-blind, split-face, trial. Wrinkle depth and volume changes were used as endpoints. Given our previous study [17] demonstrated that GHK-Cu is highly hydrophilic, a lipid-based nano-carrier system was employed for delivery into the skin. This trial is the first to investigate the biological effect of GHK-Cu formulated into a nano-carrier.

Materials and Methods

Materials

GHK-Cu was purchased from Salkat Ltd (Auckland, New Zealand). Snowberry New Zealand Limited provided the New Radiance Face Serum (NRFS - nano-carrier containing GHK-Cu in a serum vehicle) and CONTROL (the serum vehicle without GHK-Cu or nano-carrier). Strivectin SD Advanced Intensive Concentrate (SSID) was purchased online. All Taqman gene expression assay kits, reagents, 18S housekeeper genes, SuperScript complimentary DNA Synthesis kit and PureLink RNA mini kit were purchased from Applied Biosystems (Life technologies, Auckland, New Zealand). Reagents for agarose preparation (SeaKem LE agarose) were purchased from Lonza (Auckland, New Zealand). Human adult primary dermal fibroblasts (HDFa) were purchased from Invitrogen (Invitrogen, USA). All phosphate buffered saline (PBS, 10 mM) was freshly prepared (8 g NaCl, 0.2 g KCl, 1.44 g Na2HPO4, 0.24 g KH2PO4 in 1 litre water and adjusted to pH 7.4 with HCl. All water used was prepared by reverse osmosis.

Cell culture of primary human dermal fibroblasts

Fibroblasts, passage numbers between 4 and 8, were seeded in 6- well growth plates at a density of 30,000 cells per well. Cells were incubated in 3 mL DMEM with 1.5% FBS and 50 U ml-1 each of penicillin and streptomycin for 24 h at 37°C in a Heracell 150i incubator (Thermo Fisher Scientific, Victoria, Australia) maintaining a 5% CO2 atmosphere with >80% relative humidity. The cells were incubated with GHK-Cu solutions in DMEM for 24 hours. The final concentrations of GHK-Cu were 0.01, 1 and 100 nM, a range previously reported to produce a response in MMP and TIMP expression in in vitro rat fibroblasts [7]. Control cultures received an additional volume of water (without the GHK-Cu) at the same time as the treated cultures. Each condition was examined in triplicate, with each culture tested three times. Following removal of medium, cells were processed for RNA extraction.

RNA extraction and PCR

RNA extraction was performed using a RNA extraction kit based on the manufacturer’s protocol (Biocolor, Carrickfergus, Ireland). Briefly, control and treated cells were washed with PBS three times, cells transferred to RNase-free tubes and centrifuged at 2000 x g for 5 minutes to obtain a cell pellet. The supernatant was removed and 0.6 mL lysis buffer added along with 1% 2-mercaptoethanol to re-suspend the pellet. The dispersion was mixed with a 70% ethanol solution at 1:1 (v/v) and centrifuged in a spin cartridge tube and attached collection tube. The collection tube was washed twice with buffer to purify the RNA, which was eluted after a final wash with RNase-free water.

RNA quality and quantity assessment

Concentration and integrity of extracted RNA was quantified using a NanoDrop 1000 spectrophotometer (NanoDrop Technologies, USA). Purity criteria was >1.8 for 260/280 ratio (the ratio of absorbance at 260 nm and 280 nm) and >2 for the 260/230 ratio (Nanodrop technical support documentation). Confirmation of quality was by Agarose gel electrophoresis. To prepare the gel substrate, 2.6 g of SeaKem LE agarose was dissolved in 65 ml tris-borate-EDTA (TBE) buffer at 80°C, placed onto the frame, and allowed to set for a minimum of 30 minutes. TBE buffer was added, covering the gel completely, before PCR product (10 μL) was added to the wells along with 5 μL Blue Juice Loading Buffer. A 100V potential difference was placed across the gel and left for 60 minutes. The gel was stained with 100 ml TBE buffer containing 5 μL of a 10 mg mL-1 ethidium bromide (EtBr) solution. After 15 minutes the EtBr was washed off with water and imaged using a GelDoc EZ Imager (Biorad Laboratories, Auckland, New Zealand).

Reverse transcription (cDNA synthesis) and RT-qPCR

The extracted RNA was reverse transcribed using the SuperScript complimentary DNA (cDNA) Synthesis kit using the manufacturer’s protocol (Applied Biosystems). Briefly, the reaction mixture comprised 14 μL of RNA free water, 4 μL of 5× VILOTM master mix and 2 μL of 10× SuperScript® Enzyme Mix. Negative reverse transcription (RT) samples were produced as a control. The samples were incubated in an Applied Biosystems Gene Amp PCR 9700 for 10 min at 25°C, 120 min at 42°C and 5 min at 85°C, sequentially.

To quantify cDNA, real time quantitative polymerase chain reaction (RT-qPCR) was used [20,21]. RT-qPCR was performed with a predesigned mix (RTMIX) consisting of 5 μL Master Mix, 0.5 μL Gene Expression Assay, 0.5 μL 18S (a housekeeper gene), and 2 μL of RNasefree water.

The RTMIX (8 μl) was combined with 2 μL of cDNA sample and placed into a 384-well MicroAmp® plate. Each cDNA sample was prepared in triplicate and each condition was measured 3 times in 3 separate culture samples. After loading all the samples, the MicroAmp® plate was sealed and centrifuged for 1 minute at 2000 rpm. The plate was placed into a Sequence Detection System (Life technologies, Auckland, New Zealand) for DNA amplification. 6-Carboxyfluorescein (FAM) was used as the fluorophore for each Taqman gene expression probe whilst 4,7,2′-trichloro-7′-phenyl-6-carboxyfluorescein (VIC) was used for the 18S housekeeper gene [22]. Amplification consisted of four-cycle stages: 50°C for 2 minutes, 60°C for 30 seconds, 95°C for 10 minutes followed by 40 cycles of 95°C for 15 seconds and 60°C for 30 seconds. To calculate the relative gene expression the results from RTqPCR were normalized using the 2-ΔΔCt method [23].

Collagen and elastin quantification

Cells were seeded in 12-well plates (surface area 3.77 cm2 per well) at a density of 1 × 105 cells per well. Each well contained 3 mL cell culture medium without bovine serum. After the initial 24 h to allow cell settling and attachment, each well was supplemented with GHKCu solution (in water, 100 μL). Cells were treated for 48 and 96 h. Control samples were supplemented with water. Following treatment, cell culture medium was collected for collagen measurement, while the cells were detached by incubation with 250 μL of trypsin for 10 minutes at 37°C for elastin measurement.

Collagen measurement: Total soluble collagen content in culture medium was measured using the Sircol soluble collagen assay kit as described by manufacturer’s protocol (Biocolor, Carrickfergus, Ireland) [24]. To isolate the collagen, 1 mL of cell culture medium was placed into a 1.5 mL Eppendorf tube and mixed with a 200 μL aliquot of the provided isolation and concentration reagent (polyethylene glycol TRIS-HCl buffer, pH 7.6). Following vortex mixing for 30 seconds, samples were incubated at 4°C overnight to allow collagen to precipitate. Samples were then treated as per manufacturer’s instructions (Biocolor, Carrickfergus, Ireland) and transferred to a 96 well plate for absorbance measurement at 555 nm. Reagent blanks were also measured for absorbance. The concentration of collagen was then calculated using a standard curve.

Elastin measurement: The cell suspension was transferred to a microcentrifuge tube and pelleted at 12,000 × g. 1.0 M oxalic acid solution (100 μL) was added to the pellet making a final concentration of 0.25 M oxalic acid solution and heated to 100°C for 1 h to convert insoluble elastin to soluble α-elastin. The samples were then treated as per manufacturer’s instructions (Biocolor, Carrickfergus, Ireland) and transferred to a 96 well plate for reading at 513 nm [24]. Reagent blanks (250 μL) including oxalic acid, PBS and water served as controls. Absorbance at 513 nm from these control reagents was subtracted from the final readings of the sample, providing a reading for elastin. Absorbance values were converted into concentrations using a standard curve.

Effects on human facial wrinkle parameters

The trial was conducted at Dermatest GMBH, a cosmetic research institute, with 40 female volunteers (aged from 40 to 65 years) over the course of 8 weeks. All test products were supplied as identically packaged coded containers with investigators and subjects blinded to treatments. Prior to application all the subjects underwent a dermatological examination and a 10-day period of no cosmetic use. All participants gave informed consent.

Participants were randomly placed into two treatment groups: Group 1) NRFS serum, the Nano-carrier enhanced GHK-Cu serum and a product containing Matrixyl® 3000 (SSID) as a positive control, and Group 2) NRFS serum and CONTROL, the latter contained no GHK-Cu or nano-carrier, acting as a negative control. All subjects applied the NRFS serum twice each day (in the morning and again in the evening) to the right side of the face, and either the SSID (Group 1) or CONTROL (Group 2) to the left side of the face according to the same instructions. The participants were instructed to not apply other skin care products to the test areas during the course of the trial.

Measurement of wrinkle depth and volume

The Phaseshift Rapid in vivo Measurement of the Skin system (PRIMOS, GF Messtechnik GmbH, Teltow, Germany) was used as a three-dimensional analytical instrument for this study. The principle of this instrument has been previously described by Jaspers et al. [25]. Briefly, a digital stripe projection technique is used as an optical measurement process. A parallel stripe pattern is projected onto the skin over the wrinkle area and depicted on the CCD chip of a digital camera connected to an evaluation computer. The measurement head is then moved close to the immobilized head of the participant (Canfield Scientific Inc., NJ, U.S.A). The parallel projections are then distorted by the elevation differences on the skin and a threedimensional effect recorded. The distortions provide a qualitative and quantitative measurement of the skin profile. They are digitised and quantitatively evaluated using software attached to the PRIMOS system. The measurement area was a 30 × 40 mm region, located as close as possible to the corner of the eye. A single large wrinkle was identified on each subject and measured for wrinkle depth and volume at 4 (NRFS and SSID) and 8 weeks (NRFS, SSID and CONTROL). Measurements of the same wrinkle at 4 and 8 weeks after treatment were compared with initial measurements.

Data analysis of in vivo trial data

R package lme (R version 3.0.2) was used to calculate statistical significance using regression analysis. The significance level was set at 0.05. For the primary analysis, comparing the NRFS serum and SSID, a multiple linear mixed model was used with random intercepts for each patient, to take account of the grouping by participants in the data. In the multiple linear mixed model, the groups being compared (NRFS vs SSID and NRFS vs CONTROL), with the right side of subject’s face belonging to one group and the left side of the same subject’s face belonging to the other treatment were made exactly comparable with regard to starting wrinkle depth, and eliminated the individual biological variation of the subjects. The equivalent analysis was performed for wrinkle volume.

Percentages reported in this study were calculated by averaging the percentage changes for each individual in the trial. The individual changes were calculated from the relative change percentages; e.g., starting skinfold depth (833.4 μm) was compared against the 8-week depth measurement (722.1 μm). The difference (111.3 μm) was expressed as a 13.35% change.

Results

RNA quality and quantity

All samples for RNA analysis met the purity criteria confirmed by Agarose gel electrophoresis. The 260/280 ratio was 1.95 ± 0.15 and the 260/230 ratio was 2.12 ± 0.08, over all the samples examined.

Effects of GHK-Cu on mRNA expression of MMPs and TIMPs

After 24 hours, expression of MMP1, MMP2, TIMP1 and TIMP2 in the GHK-Cu treated cell lines, relative to untreated controls, all showed a concentration dependency effect (Figure 1) with increased expression at lower GHK-Cu concentrations except for the TIMP2. For MMP1 and MMP2, interestingly, only the lowest concentration (0.01 nM) treatment resulted in significantly increased expression (p=0.03). TIMP1 expression was significantly increased at all concentrations, with a concentration effect, while TIMP2 expression was significantly decreased at higher concentrations (1 and 100 nM).

Effects of GHK-Cu on collagen and elastin production

Both collagen (0.0726x+0.05, R2=0.983) and α-elastin (0.0178x + 0.05, R2=0.984) standard curves showed a linear relationship between the absorbance and concentration within the ranges tested (5-15 μg/ml and 12.5-50 μg/ml respectively).

After treated with GHK-Cu solutions, the production of collagen or α-elastin by fibroblasts only slightly increased at 48 hours compared with the non-treated cells (Table 1). For both collagen and α-elastin, GHK-Cu significantly increased secretion over the controls at 96 hours. There was an inverse dose dependent response for collagen production at 96 hours. Alpha-elastin was increased by approximately 30% at all concentrations but without a clear concentration dependency.

ProductionConcentration of GHK-Cu (nM)Treatment (hours)
04896
Collagen0 (control)6.97 ± 1.07.55 ± 0.315.29 ± 0.4
0.01 8.64 ± 0.318.04 ± 1.8*
1 8.14 ± 0.717.07 ± 1.4*
100 8.53 ± 0.516.63 ± 1.6*
α-Elastin0 (control)36.57 ± 3.879.03 ± 1.5200.4 ± 2.5
0.01 82.84 ± 4.0257.97 ± 2.5**
1 80.48 ± 5.6271.09 ± 4.3**
100 84.08 ± 2.4268.2 ± 2.6**

Table 1: Collagen and elastin levels (μg/ml) of HDFa cells treated with GHK-Cu. Data are means ± SD, n=3. * p<0.05 and ** p<0.017 compared to the control.

As the expression of MMP1 and TIMP1 reduced, with increasing concentration of GHK-Cu, there was a corresponding decrease in collagen. A similar trend did not occur for α-elastin.

Effect of GHK-Cu on wrinkle parameters

The Snowberry New Radiance Face Serum (NRFS), SSID (Strivectin SD Advanced Concentrate), and CONTROL (Control vehicle) were well tolerated by 39 of the 40 subjects throughout the eight-week application period. One participant experienced minor unwanted skin reactions on both the right and left side of their face after application of the NRFS serum and SSID. This subject ceased application and symptoms resolved without medical treatment. Wrinkle depth and volume changes at four and eight-weeks are summarised in Table 2. Comparisons and statistical significance between treatment groups are given in Table 3.

 TreatmentWrinkle parameter (weeks)Percent change from baseline 
 NRFS serumDepth (4)-18.3 ± 10.3
 (n=19)Depth (8)-26.8 ± 12.8
  Volume (4)-17.2 ± 8.1
Group 1 Volume (8)-25.8 ± 9.4
 SSIDDepth (4)-15.9 ± 8.6
 (n=19)Depth (8)-22.4 ± 8.5
  Volume (4)-16.8 ± 8.6
  Volume (8)-20.0 ± 7.8
 NRFS serumDepth (8)-20.3 ± 8.7
Group 2 (n=20)Volume (8)-24.1 ± 8.6
 CONTROLDepth (8)-15.3 ± 7.2
 (n=20)Volume (8)-15.0 ± 5.2

Table 2: Percentage changes in wrinkle depth and volume over the trial period from 39 volunteers (mean ± SD).

GroupParameterIntergroup differenceaImprovement (%)bp-value
NRFS serum versus SSIDWrinkle Depth-32.8 µm23.4%0.0577
Wrinkle Volume-0.3 m331.60%0.0044
NRFS serum versus CONTROL serumWrinkle Depth-28.3 µm32.8%0.0123
Wrinkle Volume-0.4 m355.80%<.0001

Table 3: Comparisons between treatments after 8 weeks. a The Intergroup difference is the average depth or volume change compared between the treatments within the same group. A negative result indicates that change was greater for the NRFS as compared to the other treatment. b The average improvement of NRFS compared with the other treatment within the group.

At 8 weeks the NRFS decreased wrinkle volume by 31.6% more than the SSID product (p<0.01). Wrinkle depth of the NRFS serum decreased by 23.4% more so than the SSID product (p=0.0577). The difference between the two treatments was most marked from weeks 4 to 8. SSID decreased wrinkle volume by 18.85% over the last 4-week period while NRFS serum decreased volume by 49.59% (percentage change in the mean individual percentage changes from the end of week 4 to the end of week 8) indicating that change was slowing in the SSID group at a faster rate than that of the NRFS serum. Compared to Control there was a 55.8% relative reduction wrinkle volume with the NRFS serum (p<0.01) and a 32.8% decrease in wrinkle depth (p=0.0123).

Discussion

In the present paper we report on the effects of GHK-Cu on synthesis of collagen and elastin, and expression of MMP1, MMP2, as well as tissue inhibitors of metalloproteinases (TIMP), TIMP1 and TIMP2, by human adult dermal fibroblasts (HDFa). These data are presented alongside the results of a human clinical trial investigating effects of GHK-Cu on wrinkle depth and volume.

MMPs/TIMPs expression

Application of GHK-Cu at all the tested concentrations to cultured human dermal fibroblasts increased mRNA expression of both MMP1 and MMP2. TIMP1 mRNA expression increased and to a greater extent than MMP1, suggesting net inhibition of proteolytic activity for collagens. GHK-Cu, however, did not change TIMP2 significantly at 0.01 nM, and at higher concentrations decreased expression (p<0.05) (Figure 1). The findings of increased MMPs and TIMP1 mRNA following exposure to GHK-Cu are consistent with the findings of Simeon et al. [5] who also examined effects of GHK-Cu on MMPs of rat fibroblast cells. The inverse dosage dependent response results from this study, however, do differ from a study showing that increasing the free copper concentration increases both MMP1 and TIMP1 expression [25]. Our study with GHK-Cu shows that at higher doses there is generally little effect or inhibition of both MMPs and their TIMPs. It is thus likely that GHK-Cu effects that we observed are not due to free copper.

Irrespective to the dose response, on the other hand, there was clear correlation between MMPs with their TIMP levels in the GHK-Cu treated cells; a high concentration of MMP was accompanied with a high level of its TIMP (Figure 1). A simultaneous increase and decrease of various MMPs and TIMPs has been previously reported [5]. It is important to note that TIMPs regulate the proteolytic activity of MMPs by direct interaction with these enzymes, and not by regulation at a transcriptional level [26]. It is also important to note that TIMP1 acts against all members of both collagenase and gelatinase classes [27], thus the no change, or decrease in TIMP2 at higher concentrations of GHK-Cu, does not necessarily signal a shift in favour of inflammatory changes and increased degradation. The relative increase in TIMP1 following exposure to GHK-Cu is consistent with a shift to matrix production and growth, and both TIMP1 and TIMP2 are considered to have growth factor-like functions [27].

Collagen/elastin production

Marquart et al. [4] reported a dosage dependent increase in the amount of collagen produced by human fibroblasts incubated with GHK-Cu. The response peaked at 1 nM with higher concentrations resulting in less collagen synthesized. In this present study the collagen levels were above non-treated cells at 96 hours at concentrations of 0.01-100 nM (p<0.05). Increasing GHK-Cu concentrations did not significantly increase the response. In contrast, the production of elastin, measured as α-elastin, was 30% higher than that found in the untreated cells regardless of the GHK-Cu concentrations (0.01-100 nM) (Table 1).

The increase in collagen production, albeit modest, supports the conclusion that GHK-Cu stimulates tissue growth and repair. The increase in elastin similarly supports the conclusion that GHK-Cu stimulates tissue growth and repair.

Relationship of MMPs/TIMPs expression and collagen/ elastin production

As expected MMPs and TIMPs mRNA expression, and collagen and elastin production were markedly affected by exposure of the cells to GHK-Cu. An increase of TIMP1 and TIMP2 suggested an inhibition of proteolytic activity of MMP1 and MMP2 and thus decreased fibrillar collagen (collagen) and elastin degradation which is consistent with the observed increase in collagen/elastin in this study (Table 1). Surprisingly, the increase in either collagen or elastin appeared to have little concentration-dependence on GHK-Cu although an inverse dosage dependent response with TIMP1 and TIMP2 was observed. More interestingly, although MMP2 increased and TIMP2 decreased with the treatment with GHK-Cu (0.01-10 nM), which in theory mean a decreased elastin level in the treated cells, an increase in elastin level was observed, and again with a little dose-dependence. This means single factor, either MMP or TIMP cannot determine the level of the matrix protein.

To fully understand the mRNA results, the ratios of TIMP/MMP expression were calculated (Table 4). High (>1) and relatively consistent ratios of TIMP1/MMP1 at all GHK-Cu contractions were found. This could explain the observation with cellular secretion of collagen. The low ratio of TIMP2 to MMP2 would predict a shift towards degradation. Considering however that TIMP1 acts against all members of the gelatinase classes (MMP2) and that TIMP2 also acts on MMP2, the ratio of (TIMP1+TIMP2)/MMP2 was used as a measure of prediction (Table 4).

RatioGHK-Cu concentration (nM)
0.011100
TIMP1/MMP13.762.653.1
TIMP2/MMP20.430.370.31
(TIMP1+TIMP2)/MMP21.721.391.07

Table 4: TIMP/MMP ratios on HDFa following 24 hour treatment with GHK-Cu at difference concentrations (calculated from the mean values from data in Figure 1).

Clinical study

The effects on MMP and TIMP expression with the associated increase in collagen and elastin production supported the investigation of topical GHK-Cu treatment in human volunteers. Due to the physicochemical properties of GHK-Cu hindering topical absorption, and given previous studies have evaluated topical GHK-Cu delivery in vivo [12,13], this present study used nano-carriers to facilitate delivery of GHK-Cu to the dermis. The clinical trial investigated the effect of GHK-Cu on facial wrinkle volume and depth. The application of GHK-Cu in nano-carriers (NRFS serum) resulted in a significant and improved reduction in facial wrinkle volume and depth compared to the CONTROL (serum only) and a reduction in wrinkle volume compared to the SSID, a commercially available product containing a lipophilic derivative of GHK (Matrixyl 3000®). This confirms the NRFS, containing GHK-Cu in a carrier system, is more effective than the SSID product for total wrinkle volume reduction. It also shows that improvement can be seen with continued use, observed as wrinkle depth and volume reductions between 0-4 and 4-8 weeks. The NRFS serum also significantly outperformed the CONTROL (vehicle only) in regards to wrinkle volume and depth. This evidence proves that it was the nano-carrier that impacted significantly on the total wrinkle reduction and not the formulation. Of particular importance was that all participants using NRFS showed a reduction in wrinkle volume and depth.

Importantly the nano-carrier encapsulated GHK-Cu was well tolerated and similar to SSID with only one participant reacting to both. Side-effects of anti-wrinkle agents such as tretinoin are relatively common and include peeling, dryness and erythema [28]. None of these were experienced by participants in the GHK-Cu trial, notwithstanding the aforementioned reaction.

Conclusion

GHK-Cu significantly increased cellular production of collagen and elastin by HDFa cells with little concentration dependency (0.01-10 nM). Incubation with the tripeptide also correlated with a relative higher mRNA expression of the respective TIMP(s) than the respective MMP in the cells.

Topical application of GHK-Cu with the aid of nano-carrier delivery systems reduced wrinkle volume to a significantly greater extent than the vehicle alone or a commercial product containing Matrixyl 3000®, a GHK lipophilic derivative.

Acknowledgements

Independent statistical analysis was performed by Jessica McLay based at the Department of Statistics, The University of Auckland, New Zealand.

Disclosure

This study was funded by Snowberry New Zealand Ltd in collaboration with Callaghan Innovation (Grant Number: ENDU1101.) Snowberry had no involvement in the collection, analysis, interpretation of data, decision to submit or in the writing of this article.

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Figure 1: Effect of GHK-Cu on gene expression of MMP1, MMP2, TIMP1 and TIMP2 in HDFa cultures after incubation for 24 hours. Data are means ± SD, n=3 from three individual experiments. * Denotes p<0.05 statistical difference from control (untreated cells).

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