2.1. GHK Improves Skin Regeneration

Skin’s ability to withstand damage and repair itself is highest in children and young individuals because of well-functioning repair and protective mechanisms. However, with age, skin’s ability to repair damage declines. GHK content is highest in the plasma of young, healthy individuals. At age 20, the plasma level of GHK is about 200 ng/mL (10⁻⁷ M), and by the age of 60, it declines to 80 ng/mL. In the experiment that led to discovery of GHK, plasma from young individuals added to liver tissue obtained from older individuals, caused old liver tissue to produce proteins more characteristic of younger individuals [6].

In the 1980s, Maquart et al. proposed that GHK may be an early signal for skin repair. The GHK amino acid sequence is present in the alpha 2(I) chain of type I collagen, and when damage activates proteolytic enzymes, GHK is released into the site of an injury [7]. A number of experiments established that GHK stimulates synthesis of collagen, selected glycosaminoglycans and small proteoglycan decorin [8,9]. It also modulates activity of key metalloproteinases, which are enzymes that facilitate breakdown of proteins of extracellular matrix, as well as activity of anti-proteases. This suggests a general regulatory effect on protein breakdown in skin, helping to prevent both buildup of damaged proteins and excessive proteolysis [10,11]. Since excessive breakdown of the dermal matrix as well as inadequate removal of damaged proteins can negatively affect skin’s health and appearance, GHK’s ability to regulate both metalloproteinases and their inhibitors can support skin regeneration and improve its appearance.

GHK also demonstrated beneficial effects on skin fibroblasts, which are considered key cells in the skin regeneration process. Fibroblasts not only synthesize structural elements of the dermal matrix but also produce a wide range of growth factors essential for skin repair. GHK, in combination with LED irradiation (light emitting diode irradiation, 625–635 nm), compared with the LED irradiation alone increased: cell viability 12.5-fold, production of the basic fibroblast growth factor (bFGF), 230%, and collagen synthesis, 70% [12].

GHK-Cu has been found to stimulate epidermal basal cells, markedly increasing integrins and p63 expression. The cells’ shape became more cuboidal, which indicates an increase in their stemness [13].

2.2. Cosmetic Use of GHK-Cu

A number of clinical studies confirmed GHK-Cu’s ability to improve appearance of aging skin. A facial cream containing GHK-Cu applied for 12 weeks to the facial skin of 71 women with mild to advanced signs of photoaging increased skin density and thickness, reduced laxity, improved clarity, reduced fine lines and the depth of wrinkles [14].

A GHK-Cu eye cream applied for 12 weeks to around-the-eye area of 41 women with mild to advanced photodamage performed better than placebo and vitamin K cream. It reduced lines and wrinkles, improved overall appearance, and increased skin density and thickness [15].

GHK-Cu applied to thigh skin for 12 weeks improved collagen production in 70% of the women treated, in contrast to 50% treated with the vitamin C cream, and 40% treated with retinoic acid [16]. In addition to improving skin laxity, clarity, firmness and appearance, reducing fine lines, coarse wrinkles and mottled pigmentation, and increasing skin density and thickness, GHK-Cu cream applied twice daily for 12 weeks also strongly stimulated dermal keratinocyte proliferation [17].

With their pilot study for topical application of copper tripeptide complexes in aged skin, Krüger et al. confirmed an increase in skin thickness in the range of the epidermis and dermis, improved skin hydration, a significant smoothing of the skin by stimulating collagen synthesis, increased skin elasticity, a significant improvement in skin contrast and an increased production of collagen I [18,19].

GHK-Cu at 0.01, 1 and 100 nM incubated with human adult dermal fibroblasts increased production of elastin and collagen. GHK also increased gene expression of MMP1 and MMP2 at the 0.01 nM. All concentrations increased TIMP1. The effects of GHK-Cu were also investigated in a randomised, double–blind clinical trial. Female volunteers applied GHK-Cu, encapsulated in nano-lipid carrier twice a day in the course of 8 weeks using either carrier alone or the commercially available peptide Matrixyl^® 3000 as controls. Compared to Matrixyl^® 3000, GHK-Cu produced a 31.6% reduction of wrinkle volume. Compared to control serum, GHK-Cu reduced wrinkle volume 55.8% and wrinkle depth 32.8% [20].

2.3. Animal Studies Confirm Wound Healing Activity of GHK

Multiple animal studies have established the wound healing activity of GHK. It appears that GHK stimulates wound healing through a variety of mechanisms. In rabbit experimental wounds, GHK alone or in combination with high dose helium–neon laser improved wound contraction and formation of granular tissue, as well as increasing activity of antioxidant enzymes and stimulating blood vessel growth [21,22]. Collagen dressing with incorporated GHK (PIC-Peptide Incorporated Collagen) accelerated healing of wounds in healthy and diabetic rats. The treated group displayed higher glutathione (GSH) and ascorbic acid levels, better epithelialization, as well as increased synthesis of collagen and activation of fibroblasts and mast cells in wounds. In healthy rats, treatment of wounds with PIC increased collagen 9-fold [23,24]. GHK-Cu improved healing of ischemic open wounds in rats. Wounds displayed faster healing, decreased concentration of metalloproteinases 2 and 9 as well as of TNF-β (a major inflammatory cytokine) compared with vehicle alone or with untreated wounds [25].

One problem with GHK-Cu is that it is very sensitive to breakdown by carboxypeptidase enzymes. Wounds such as diabetic skin ulcers or bedsores usually develop a “wound serum”, thought to be generated by airborne bacteria settling on the wound. The “serum” rapidly breaks down GHK and probably other growth factors such as TGF (Transforming Growth Factor) and PDGF (Platelet Derived Growth Factor).

2.4. Stimulation of Blood Vessel and Nerve Growth

Nerve and blood vessel growth is an important factor in skin healing and regeneration.

Sage et al. observed that GHK and related peptides are produced in the course of protein breakdown after an injury from a SPARC protein. SPARC (Secreted Protein Acidic and Rich in Cysteine) is a glycoprotein, mostly expressed in embryonic tissues and in tissues undergoing repair and remodeling. At initial stages of tissue repair, GHK and other peptides containing the GHK sequence (such as KGHK), which are released from SPARC in the course of proteolysis, stimulate new vessels growth. Later in the healing process, GHK and GHK-related peptides inhibit blood vessel growth [26].

2.5. Anti-Oxidant and Anti-Inflammatory Actions

As animal experiments show, treatment of wounds with GHK leads to elevated levels of antioxidant enzymes. GHK also possesses strong antioxidant and anti-inflammatory actions. GHK inactivated damaging free radical by-products of lipid peroxidation, such as 4-hydroxynoneal, acrolein, malondialdehyde, and glyoxal, protecting cultured skin keratinocytes from ultraviolet (UV)-radiation [31]. GHK was shown to completely block Cu(2+)-dependent oxidation of low density lipoproteins (LDL). Another well-known anti-oxidant, which is also widely used in skin care, superoxide dismutase (SOD1), gave only 20% protection [32]. GHK also prevents damaging effects of lipid peroxidation, by binding its by-products such as acrolein and 4-hydroxynonenal [33,34].

GHK:Cu(2+) reduced iron release from ferritin by 87%. Ferritin in blood plasma can store up to 4500 atoms of iron per protein molecule, which is a well-known catalyst of lipid peroxidation—a chain reaction, which produces a slew of free radicals, leading to DNA, protein and cell membrane damage. Disturbances in iron metabolism contribute to many pathological conditions, including brain damage and neuron death under various neurological conditions. When iron is released from ferritin, it can form an Fe(2+)/Fe(3+) complex and start the chain reaction of lipid oxidation [35].

2.6. Lung COPD and Acute Lung Injury

Successful tissue regeneration requires collaboration of multiple cells, which is orchestrated by various cytokines and growth factors. This means that in order for regeneration to be successful, these molecules have to be produced and released in the right amount and in the right place. This is important because no signal molecule works on its own, but instead engages in a crosstalk, which leads to activation of certain cellular pathways. Among pathways involved in skin regeneration are cellular pathways regulated by TGF-β and integrins [36].

GHK appears to support remodeling and restructuring of connective tissue, modulating expression of numerous genes, including up-regulation of genes of the TGF-β pathway. This is evident from a study which demonstrated GHK’s ability to reverse expression of key genes, included in a gene signature of COPD—Chronic Obstructive Pulmonary Disease. The expression of 127 genes was altered in COPD patients. More severe emphysema symptoms were correlated with the degree of change in gene expression. Genes whose expression was associated with inflammation were upregulated, and genes involved in tissue remodeling and repair were downregulated significantly. Using the Connectivity Map, a software gene profiling tool developed by the Broad Institute, the researchers sorted through gene expression profiles of biological molecules and came up with GHK as a compound which could reverse changes in gene expression associated with emphysematous destruction, such as decreased activity of genes involved in the TGF-β pathway. GHK was able to change the gene expression pattern to its opposite—activation of the TGF-β pathway.

GHK was then tested in vitro to confirm its positive effects on connective tissue. Lung fibroblasts from COPD patients, which had impaired ability to contract and restructure collagen, were treated with GHK or TGF-β. Both molecules restored function of fibroblasts. They also had an elevated expression of integrin beta 1 [37,38].

The use of GHK-Cu in mice protected their lung tissue from induced acute lung injury (ALI) and suppressed the infiltration of inflammatory cells into the lung. The GHK-Cu also increased superoxide dismutase (SOD) activity while decreasing TNF-1 and IL-6 production through the blocking activation of NFκB’s p65 and p38 MAPK (mitogen activated protein kinase). Mitogen activated protein kinases are kinase enzymes that play crucial part on cellular signaling. P38 MAPK pathways enable cells to respond to a wide range of external stressors, and affect skin differentiation, apoptosis, mobility and gene expression. NFκB p65 activation has been found to be correlated with many diseases of aging and cancer development [39].

2.7. Blocking of Cortisone Effects

Topical steroids, such as cortisone, are often prescribed for treatment of inflammatory cutaneous disorders, can inhibit wound repair, as well as produce skin thinning and other defects [40].

GHK-Cu, administered systemically to mice, rats, and pigs, has protective effects on cortisone—induced inhibition of wound healing [41].

2.8. Suppression of Fibrinogen

GHK was isolated as a plasma molecule that suppressed fibrinogen. Fibrinogen is best known for its ability to form blood clots. However, it also heavily influences the flow of blood through the microcirculation, where blood acts as a thixotropic fluid, somewhat like toothpaste, flowing under increased pulses of blood pressure, then reverting to a semi-solid gel. Elevated fibrinogen greatly increases the blood viscosity in the microcirculation by increasing red blood cell “stacking” or rouleaux formation. Studies in Germany and Scotland have found that fibrinogen levels are the top risk factor for cardio vascular diseases (CVD). The Prospective Cardiovascular Münster (PROCAM) study followed 5389 men for 10 years. It found that the incidence of coronary events in the top third of the plasma fibrinogen levels was 2.4-fold higher than in the bottom third. Individuals in the top third of levels of low-density lipoprotein (LDL) cholesterol who also had high plasma fibrinogen concentrations had a 6.1-fold increase in coronary risk. Unexpectedly, individuals with low plasma fibrinogen had a low incidence of coronary events even when serum LDL cholesterol was high [42].

The Scottish Heart Health Study followed 10,359 men and women for 2 years, and fibrinogen was the single most powerful risk factor for CVD risk or death and more predictive than lipoprotein cholesterol. The increase in (relative risk) between the highest and lowest fibrinogen levels was 301% for men and 342% for women (CVD death) and 259% for men and 220% for women (death from any cause) [43].

As mentioned above (Park et al.), GHK suppresses the production of Interleukin-6, a main positive regulator of fibrinogen production, both in cell cultures and in mice. As our gene profiling data indicate, GHK downregulates (−475%) the gene for the beta chain of fibrinogen. Since equal amounts of all three polypeptide chains are needed to produce fibrinogen, when synthesis of one of the chain of fibrinogen is suppressed, it will have a general inhibitory effect on fibrinogen synthesis [44].

2.9. Skin Remodeling and Anti-Cancer Actions

A major concern for any substance, which activates cell growth and tissue remodeling—is whether it can also trigger cancer. Therefore, it is very important to notice that GHK, which repairs skin, also possesses potent anti-cancer properties.

In 2010, Hong et al. used Broad Institute’s Connectivity Map to find molecules that could inhibit metastatic colon cancer. The Connectivity Map contains expression profiles that were evaluated in five cancer cell lines, in response to 1,309 bioactive small molecules. A search through the database produced only two substances that were able to down-regulate expression of “metastatic” genes— two skin remodeling substances, GHK and the plant alkaloid, securinine. GHK produced the result at a low non-toxic 1 micromolar concentration and securinine at 18 micromolar. GHK suppresses RNA production in 70% of 54 human genes overexpressed in cancer patients, including “node molecules” YWHAB, MAP3K5, LMNA, APP, GNAQ, F3, NFATC2, and TGM2. These molecules play key roles in regulation of important molecular pathways [45]. This shows that GHK is involved in gene regulation of various biochemical pathways, and it seems to be resetting the gene activity back to health, which leads to the improvement of tissue repair [46].

UV-radiation and other damaging factors can damage skin cells’ DNA, which can potentially lead to skin cancer. One of the main protective mechanisms is apoptosis or programmed cell death. Normal healthy skin cells have checkpoint systems to self-destruct if they are synthesizing DNA incorrectly. When apoptosis is inhibited, skin cancer risk greatly increases. Also, apoptosis is the mechanism through which many anti-cancer treatments, including melanoma treatments, work. Some common cosmetic ingredients, such as elderberry extract, can enhance effectivity of cancer treatment by enhancing apoptosis in malignant cells [47].

Matalka et al. demonstrated that GHK, at 1 to 10 nM, inhibited the growth of human SH-SY5Y neuroblastoma cells and human U937 histiocytic lymphoma cells. It also re-activated the apoptosis system, as measured by the caspases 3 and 7. In contrast, GHK stimulated the growth of healthy human NIH-3T3 fibroblasts [48].

In 1983, using a method developed by Linus Pauling’s group [5], we tested the mixture of GHK-copper 2+ plus ascorbic acid (vitamin C) on the growth of sarcoma-180 in mice. This gave a very strong suppression of the cancer. These results remained unpublished until 2014, when we could supplement these findings with gene data. We used the Broad Institute Connectivity Map to investigate the effect of GHK on genes relevant to cancer growth. GHK upregulated the expression of 10 caspase and caspase-associated genes. It also affected 84 genes associated with DNA repair and other processes, relevant to anti-cancer effects. The anti-cancer activity of GHK may be linked to its known tissue remodeling effects [49].

2.10. Ubiquitin Proteasome System

The ubiquitin proteasome system (UPS) is a system that processes and clears damaged proteins. When this system is not functioning properly, damaged proteins may start accumulating. Aging is associated with decreased activity of the ubiquitin proteasome system. So far, there are no effective therapies to increase the UPS activity. Recent work has demonstrated that proteasome activation by either genetic means or use of compounds slows down aging [50].

We performed a search, using gene titles containing “ubiquitin” or “proteasome”. GHK strongly stimulates the gene expression of the UPS system, increasing activity of 41 genes and suppressing 1 gene. See Table 3.

According to Broad Institute data, UPS genes changed at least 50% UP or DOWN. GHK increased gene expression in 41 UPS genes while suppressing 1 UPS gene. This should have a positive effect on this system.

2.11. Anti-Pain, Anti-Anxiety, Anti-Aggression

GHK has potent anti-pain, anti-anxiety (anxiolytic) and anti-aggression actions.

Anti-pain effects were measured by determining how long it took for mice to lick their paws after being placed on a mildly-hot plate. Here, GHK reduced pain at a dose of 0.5 milligrams/kilogram. GHK has a physical structure similar to cimetidine which is often used to reduce pain in humans [51,52].

The anti-anxiety, anti-pain and anti-aggression actions were found in rats. When rats are afraid, they try to hide. But within 12 min of intraperitoneal injection of GHK at 0.5 micrograms/kilogram into rats in a testing cage built as a maze, the amount of time the rats spent exploring more open and lighted areas of the maze increased, and the time spent immobile (the freeze reaction) decreased, which indicated a reduction of fear and anxiety [53]. The same occurred in a test “open field”, where the rats spent less time hiding and more time exploring the area [54].

Likewise, the anti-aggression actions were found in rats. For the experiment, two rats are placed in a small cage and then given small electrical shocks, which produces anger in the rats and led to physical attacks on the other rat. The attacks were reduced 5-fold, when rats were placed into an agression-stimulating environment 12 min after the injection of 0.5 micrograms GHK per kilogram of body weight. If scaled up for human weight, this suggests that a similar effect might be induced in humans by 35 micrograms of GHK, which is a very low and safe dosage [55].

Studies of GHK passage through the skin by Howard Maibach’s laboratory suggest that it may be possible to easily pass an adequate amount of GHK-Cu through the skin to reduce anxiety, and possibly pain [56].

A manual search of genes affected by GHK found that seven anti-pain genes increased and two genes decreased. The results are presented in the Table 4.

Psychological stress is an adaptive response that can be deleterious under certain conditions. Stress and anxiety delay epidermal barrier recovery, impair skin immune function, increase inflammation and oxidative stress. It causes activation of the hypothalamus-pituitary-adrenal (HPA) axis and the sympathetic nervous system. Skin is affected by molecules that are released during stress, such as neuropeptides, hormones, and cytokines [57]. This makes analgesic and anxiolytic effects of GHK an important part of its skin-related effects. Studies show that reducing anxiety and psychological stress can have a positive effect on chronic skin conditions such as psoriasis and atopic dermatitis [58]. Chronic stress can also impede wound healing, affecting recovery from plastic surgery and other invasive cosmetic procedures [59,60]. We believe that the GHK molecule evolved as the first response to an injury, and as such it is not surprising that it possesses analgesic and anxiolytic effects. Lowering the level of stress-related hormones and cytokines could help animals reduce inflammation and increase the chance of surviving the injury.

2.12. GHK Formulation and Delivery

GHK-Cu can penetrate the stratum corneum, which ensures its activity in cosmetic formulations [61,62]. However, to increase GHK’s penetration, it is advisable to use liposomes, including nanosized liposomes [63].

GHK is safe and very inexpensive. It can be easily incorporated into skin protective formulations, such as sunscreens and daytime creams and serums, as well as anti-wrinkle formulations. Because of its ability to improve wound healing, it is recommended after plastic surgery, chemical peels, dermabrasion, laser treatment, and so on. It will be very useful in a clinical setting and in assisted living senior facilities as a wound dressing, especially for diabetic and ischemic wounds.

Its safety record and its ability to reverse gene expression back to health warrant its use as a dietary supplement to support health and vitality of skin, hair and the entire body.