Human skin penetration of a copper tripeptide in vitro as a function of skin layer
Objective and design
Skin retention and penetration by copper applied as glycyl-l-histidyl-l-lysine cuprate diacetate was evaluated in vitro in order to assess its potential for its transdermal delivery as an anti-inflammatory agent.
Materials and methods
Flow-through diffusion cells with 1 cm2 exposure area were used under infinite dose conditions. 0.68% aq. copper tripeptide as permeant was applied on isolated stratum corneum, heat-separated epidermis and dermatomed skin and receptor fluid collected over 48 h in 4 h intervals using inductively coupled plasma mass spectrometry to analyze for copper in tissues and receptor fluid.
The permeability coefficient of the compound through dermatomed skin was 2.43 ± 0.51 × 10−4 cm/h; 136.2 ± 17.5 μg/cm2 copper permeated 1 cm2 of that tissue over 48 h, while 97 ± 6.6 μg/cm2 were retained as depot.
Copper as tripeptide was delivered in potentially therapeutically effective amounts for inflammatory disease.
Acute and chronic inflammation are characterized by changes in the metabolism of copper and by a pronounced responsiveness to therapy with copper compounds [1, 2]. The copper content and ceruloplasmin activity of serum are significantly elevated above normal values in inflammatory diseases in man and laboratory animals, and copper in widely different chemical forms is used as therapeutic agent in therapy for chronic and acute inflammation . The rise in total serum copper measured in inflammation is held to represent the natural anti-inflammatory (AI) response of the organism itself. The responsiveness of inflammatory disorders to copper supplementation noted suggests that the control exerted by endogenous copper on inflammation is susceptible to enhancement by exogenous sources. When copper complexes were administered subcutaneously, the reduction in inflammation was proportional to total amount of copper injected .
Copper derivatives of well-known antiarthritic drugs, including steroidal and nonsteroidal AI agents. The copper complexes of those drugs have been found to be active or more effective AI agents than the parent drugs .
The AI effect of copper is non-specific with respect to inflammatory pathologies and is enhanced by exogenous sources .
AI activity of exogenous copper sources is independent of ligand . On topical administration, copper complexes are subject to dynamic ligand exchange in the process of diffusion through skin . Thereby, copper oxidation state can change from monovalent to divalent to the elemental state, and the original complex administered topically is likely to differ from the complex which subsequently forms with endogenous amino acids to act as an AI agent .
The function of copper ligands thus is to promote skin penetration only, and this study evaluates skin penetration of a nature-identical copper complex . If therapeutically significant, that is, if transdermal AI therapy can be achieved, it may present an alternative to current approaches involving intra-articular or intramuscular injection, which can cause patient discomfort, particularly in arthritic patients where long term therapy is employed.
Materials and methods
The permeant was GHK-Cu received from the ProCyte Corp. (Kirkland, WA). It was not analyzed for stoichiometric purity since, in several studies, copper complexes in a physiological environment were subject to de-complexation and rearrangement, depending on pH and complex stability constants [11, 19–21]. Those investigations indicated the presence of multiple species for GHK-Cu (e.g., tri-and hexa-peptide) in solutions, together with copper-free oligopeptide. Copper as a transition metal of variable oxidation states has the ability to form complexes with different geometries and stability. Here, the copper tripeptide is summarily referred to as GHK-Cu. Concentrations are reported in terms of copper per ml of solution.
Copper penetration was studied using split thickness skin, isolated stratum corneum and entire epidermis.
Human cadaver skin was obtained from the Northern California Transplant Bank (San Rafael, CA) and stored in a freezer (−20°C) until use. All experiments were performed within 2 months after freezing. All subjects were Caucasian, aged 21–53 years, and both genders were represented.
Split thickness skin (entire epidermis with a portion of the dermis) was obtained by use of a dermatome targeted to 500 μm and stored (−20°C) until use.
Entire epidermis, (isolated. epidermis incl. stratum corneum), was obtained by heat separation (60°C) and stored (−20°C) until use.
Isolated stratum corneum was obtained from isolated epidermis which was placed on Whatman paper soaked in a 0.05% w/w trypsin Type III (from bovine pancrease, Sigma Chemical, St. Louis, USA) solution in 0.15 M phosphate buffered saline (PBS, pH 7.4, Sigma) for 2 h at 37°C. Remaining trypsin activity was blocked by submerging the stratum corneum sheet in 0.05% w/w trypsin inhibitor Typed II (from soybean, Sigma Chemicals, St. Louis, USA), then the stratum corneum sheet was washed twice in purified water, dried and stored above silica gel at room temperature.
Separation of epidermis (incl. stratum corneum) from dermis following split thickness skin diffusion experiment C (sep. epidermis): recovered split thickness skin was placed on polyethylene film, covered with polyethylene film and placed under an aluminum block heated to 65°C for 90 s, allowing separated epidermis to be lifted from the dermis.
Replicates were run on tissue from the same donor. Glass low-volume flow cells with 1 cm2 exposure area were kept at 37°C. Receptor fluid was collected over 48 h in 4 h intervals. Cells were mounted in the wells of an aluminum cell holder (Stratacor, Richmond, CA), whose internal channels were perfused with water at 37°C from a circulating water bath, and holders were mounted on a fraction collector. Water containing 1 ml/l of gentamycin sulfate solution (l00 mg/ml) was pumped through the cells at approximately 2 ml/h. Tissues were cut into circles with a biopsy punch (1 cm2), mounted on the diffusion cells and the donor chambers clamped in place.
To test for tissue integrity, prior to the experiment with tissue discs clasped in place, the peristaltic pump was run at 10 rpm, generating back pressure in the receptor cells (five times the normal pressure) for 2 h to check for leakers, which were eliminated.
About 1 ml of test solution was placed in the donor chambers, closed with ground glass stoppers and sealed with Parafilm to prevent donor solution evaporation.
At completion, the content of donor cells were collected with a pipette, the skin surface washed (3 × 2 ml) with distilled water and the rinses saved in LSC vials with polyethylene capliners (Perkin-Elmer, Shelton, CT).
For original copper levels, portions of isolated stratum corneum, isolated epidermis, as well as split thickness dermis were analyzed to determine the increment retained there during diffusion. Following diffusion experiment C, split thickness skin was also separated into epidermis and dermis (entire separated epidermis and separated dermis) for analysis of tissue depot formation in the experiment.
Experimental details and results are reported in terms of copper, analyzed by inductively coupled plasma mass spectrometry (ICP-MS).
Analyses were performed by DuPont Analytical Solutions, Wilmington, DE. There, samples were prepared by diluting 1:10 (volume/volume), using 2% HNO3 immediately prior to analysis by ICP-MS. The ICP-MS equipment used was an Agilent 7500c, operated under helium collision gas mode. The instrumental detection limit was <100 pg/ml copper. Standard solutions were prepared by diluting a 1,000-μg/ml Cu stock solution with 2% HNO3. Two separate (low concentration and high concentration) six-point external calibration curves (r2 > 0.999) were used for quantitation of both copper isotopes (63 and 65 Cu) with 89Y used as an internal standard. The low concentration calibration curve was constructed from 1, 2, 5, 10, and 20 ng/ml Cu standards and the high concentration calibration curve was from 20, 100, 1,000, 5,000, and 10,000 ng/ml Cu standards. The instrument was calibrated using the five standards above, and the samples bracketed between the standards without extrapolation either below the calibration curve or above it.
Kp (cm/h) was calculated from steady-state flux Jss (μg/cm2/h) of copper. Tissue copper levels reported from diffusion experiments were corrected for endogenous levels determined prior to the experiments.
Standard deviations (SD) were calculated by the XL program.
Copper skin penetration as function of skin strata from aq. GHK-Cu solution
Kp × 104 (cm/h) ± SD
Flux (μg/cm2/h) ± SD
Lag time (h)
Isolated stratum corneum (n = 3)
55.9 ± 13.2
38 ± 9
Isolated epidermis (n = 3)
0.003 ± 0.0006
0.002 ± 0.0004
Dermatomed skin (n = 8)
2.5 ± 0.51
1.7 ± .35
Human skin penetration and retention of copper in function of skin strata after 48 h (μg/cm2; percent of applied dose)
Isolated stratum corneum (n = 3)
(79 ± 13)
290.3 ± 69 (4.0 ± 0.15)
2,110 ± 589 (19.85 ± 3.9)
(98 ± 27)
Isolated epidermis (n = 3)
(94 ± 12)
59.7 ± 13.1 (1.0 ± 0.3)
0.60 ± 0.21 (0.006 ± 0.001)
(95 ± 12)
Split thickness (n = 8)
91 ± 14
1.0a ± 0.4 (66.0 ± 6,6)
0.09 ± 0.01 (30.9a ± 6.8)
136.2 ± 17.5 (2.0 ± 0.31)
(94 ± 7)
Copper reservoir in skin layers versus original levels from 0.68% aq. GHK-Cu at 48 h
Cu at 0 h (μg/cm2)
Cu at 48 h (μg/cm2)
Increase vs. 0 h
Isolated SC (Exp. A)
0.68 ± 0.28; n = 3
290 ± 69; n = 3
Isolated Epi (Exp. B)
0.37 ± 0.17; n = 3
61.1 ± 7.0; n = 3
Dermatomed skin (Exp. C)
1.72 ± 0.47; n = 8
53 ± 8.1; n = 8
0.37 ± 0.7; n = 8
66.7 ± 7.89; n = 8
1.72 ± 0.27; n = 8
27.63 ± 8.83; n = 8
This study evaluated absorption and skin retention of a copper tripeptide on separate skin compartments: the stratum corneum, epidermis and split thickness skin.
The amount of copper deposited from the tripeptide in the stratum corneum as well as in the entire epidermis and dermis appears remarkable. For instance, an about 400-fold increase over baseline was found in the stratum corneum alone. Such high tissue retention of copper may be attributed to intradermal decomplexation and re-binding to various endogenous amino acids with higher nucleophilic donor capacity than the original ligand.
Whether or not skin levels retained in the stratum corneum and epidermis/dermis in the diffusion process of drugs should be included in the overall estimation of absorbed material, i.e., assessing whether permeant retained in skin tissues eventually will become available systemically, is an issue of ongoing discussions. Reservoir formation in the course of in vitro studies, in particular, has been addressed . Retained drug may be lost to natural desquamation, limiting its eventual systemic availability, to be sure. On closer analysis, however, it was also found that the fraction of chemical that sloughs off versus the part that becomes systemically available depends on the ratio of lag time to the turnover time for the stratum corneum . Accordingly, at least 80% of the amount in the stratum corneum will become available if the lag time for penetration through the stratum corneum is less than about 16 h, which corresponds to 5% of the stratum corneum turnover time of typically 14 days. Consequently, based on our results where lag times ranged from 2 to 7 h, the copper retained in the stratum corneum may become largely systemically available. As a result of the experiment with dermatomed skin, by topical application of GHK-Cu under the present conditions 200–250 μg/cm2 copper would become systemically available. Since the experiment was conducted ex vivo, it is possible that even larger amounts will be retained in the deeper dermal tissue there, followed by penetration in vivo.
The permeation rate of GHK-Cu through stratum corneum seems high, the highest of all three skin strata studied. It seems consistent with a model of porous transport, as appendageal penetration is considered to be one, if not the predominant pathway for the diffusion of multipolar ions [24–26]. In addition, the high penetration level through stratum corneum alone can be explained by the fact that there was no tissue retention from underlying skin that may have limited penetration.
The high hydrophilicity of the copper tripeptide (pseudo log Pow = −4.5, quotient sat. n-octanol/sat. aqueous solution),1 is reconcilable with shunt penetration typical for highly hydrophilic solutes such as salts. Significant penetration through appendages can occur within minutes following exposure, in contrast to non-appendageal pathways [27–30].
However, the Kps of GHK-Cu through epidermis and dermatomed skin were lower at about one order of magnitude. Restriction of the pore area may cause the discrepancy in the diffusion constant. This suggests, in fact, that the diffusion pathway for copper penetration is restricted as compared to stratum corneum alone.
Skin retention and permeation by metal complexes is not often investigated, and our observations on the subject of a copper tripeptide merit more detailed discussion.
Copper is an essential trace element, critical for a variety of biological processes; for hemoglobin synthesis, enzyme activation, (i.e., superoxide dismutase), or more generally as a key component of mitochondrial, cytoplasmic and nuclear enzyme systems .
It is a key factor in the production of collagen and elastin [11, 12, 18], and the AI activity of numerous copper complexes is well documented [13, 32]. Such activity was greater than either treatment with inorganic copper salts or the complexing agents alone: while many among the parent compounds or ligands were inactive in test models of inflammation even at larger screening doses, in coordination with copper they were active. The compounds thus investigated include copper complexes of well-known antiarthritic drugs, including steroidal and nonsteroidal anti-inflammatory drugs: niflumic acid, D-penicillamine, hydrocortisone, dexamethasone, dimethylsulfoxide, clopirac, ketoprofen, (+)-naproxen, indomethacin, mefenamic acid, diclofenac, ibuprofen among a number of others. The copper complexes of those drugs have been found to be more effective anti-inflammatory agents than the parent drugs [33, 34].
Acute or chronic inflammation induces a net increase of total copper in blood, synovial fluid and inflamed areas, mobilized from endogenous stores . Chronic inflammation may lead to depletion of copper stores, implying the need for exogenous copper supply in order to effectively cope with the inflammatory pathologies. While copper complexes are commonly administered by injection, the AI activity of subcutaneous administration of copper complexes in animals was also shown to be proportional to total amount of copper injected .
The present study indicates that topical administration of copper in form of the tripeptide may offer an effective alternative to injection. Since a realistic dimension for topical application of GHK-Cu as patch may cover 10 cm2 of skin, based on our results 2.33 mg of copper would become available over 48 h. This may be compared to a subcutaneous injection of 300 mg/kg of copper aspirinate in rats without adverse effects, while it proved effective in treating experimental subplantar edema even at a dose of 10 mg/kg.
When administering copper to the organism, its toxicity should be addressed.
There is ample evidence that the copper ions released will be subject to the highly efficient homeostatic control in eukaryotic organisms that prevents its circulation at untoward levels. Levels of free ionic Cu, a relatively toxic metal, are moderated to the minimum levels sufficient for physiologic needs, reported in the range of 10−11 to 10−19 mol estimated in plasma, by binding to ceruloplasmin and metallothionein [35, 36]. The dynamic equilibrium between ceruloplasmin and metallothionein stored in liver tissue prevents toxic accumulation of free copper ion in mammals . That hepatic reservoir is released slowly back into the blood as ceruloplasmin and copper metallothionein in response to inflammation due to injury or arthritis . The average level of bound copper in human control plasma is 1.12 μg/ml ; in arthritic patients it is 1.40 μg/ml . A therapeutically effective copper level in inflammation cannot be assessed in definite terms, however, because copper status induced in response to inflammatory diseases in humans varies depending on target organ .
In contrast to injection, in topical application the skin forms a natural barrier which controls drug release over an extended period, under approximately steady conditions over a period of days. Flux, and not elimination from the skin surface seems to be the limiting step, and repeated dosage may result only in a build-up on the surface, possibly without significantly affecting the kinetics.
Based on the observation that topical applications of copper salicylate produced anti-inflammatory effects in humans volunteers, in certain countries (e.g., Australia), ethanolic Cu(II) salicylate-containing preparations (Alcusal) are available over the counter for human use to alleviate arthritic pain , an example of synergism between Cu (II) and an AI drug.
A chlorophyllin copper complex (CCC), derived from chlorophyll by replacing the chelated magnesium with copper, has anti-inflammatory and antimicrobial properties, as well as a marked stimulating effect on epithelial cell growth rates and cell regeneration. First established in tissue culture studies, these findings were confirmed clinically through wound healing and deodorizing characteristics observed in animals and humans . Administered orally, CCC is classified as a safe and effective internal deodorant by the US FDA .
In order to document the tolerability of metallic copper which must be oxidized in contact with the skin in order to penetrate, Gorter examined absorption and tolerance of metallic copper-containing ointments on human volunteers over a period of several weeks. Copper analysis in serum showed that levels had increased significantly above control levels, without overt untoward effects, either in the skin or systemically, following application of ointments containing up to 20% of the metal in its elemental form . It is well established that the Kp of organic chemicals is determined by their lipophilicity and size . This is also likely to apply to (transition) metals complexed with ligands subject to changes in pH and other parameters in the skin with depth , which determine ionization as well as availability of ligands and lipophilicity. Copper, a transition metal of variable oxidation state, has the ability to form complexes with different geometries and stability.
Similar to most transition metals, the mechanism by which copper complexes permeate skin is little understood. While copper distribution in the tissues analyzed resulted in remarkably high stratum corneum retention, through that barrier it also reached the highest values in the receptor fluid. Under present study conditions, significant passive transport of the copper species across separate skin strata of differing barrier properties seems to indicate the various potential routes of passage from the surface of the skin into the subepidermal or dermal tissue: the appendageal, the inter- and the intra-cellular pathways. Further, an additional factor appeared to determine the results obtained: intrinsic complex rearrangement in the various skin compartments leading to retention and diffusion of copper ion as a function of ligand. An undetermined part of copper might transit through the skin as the undissociated original tripeptide, and another might diffuse bound to endogenous amino acids of higher complex stability constants such as the histidinate , the predominant copper complex in plasma, with cystinate as the secondary ligand.
Studies with labeled ligands (i.e., radio-labeled) may help with further understanding the complex skin penetration behavior of copper complexes. In the present study, only diffusion followed by copper analysis on isolated or separated tissue strata allowed a measure of insight.
Quantitative assessment of skin penetration and specifying the actual penetration mechanism for a copper peptide is difficult due to possible ligand exchanges during diffusion. The results described here, however, show that a topical organic copper complex such as GHK-Cu may be an alternative to copper delivery by injection, also potentially providing a skin reservoir likely to become available over time. This effect warrants further development towards transdermal delivery of the AI agent, especially when considering GHK, a nature-identical carrier tripeptide . Several laboratories in the past had demonstrated local and systemic AI activity of copper compounds by oral, ip, iv, and particularly subcutaneous administration. Having shown permeation of the skin barrier by GHK-Cu in a therapeutically effective dose here, application of the principal to transdermal dosing now may become a realistic alternative to current, traditional routes and agents, however. Taking our results a step further, controlled release of copper for AI therapy using patch technology could be optimized with well-designed formulations. The fact that a deficit in that essential element may be significantly corrected by choosing appropriate cupriphores is a significant outcome of our endeavours towards alleviating rheumatoid arthritis and similar musculoskeletal conditions often afflicting the general population.
RCC, 2002. Determination of the partition coefficient (n-octanol/water) of copper tripeptide-1 (PC 1044). RCC Ltd, Environmental Chemistry & Parmaceutical Division; CH-4452 Ittingen/Switzerland.
We express our appreciation for the guidance received from the late Roberto Milanino, MD, of the University of Verona, who brought insight into the biological relevance of our research and to whose memory we dedicate this manuscript. We thank Len Patt of the ProCyte Corporation (Redmond, WA) for making a complimentary sample of the copper tripeptide available for our investigation. We thank Larry Ryan, DuPont Analytical Solutions (Wilmington, DE) for supervising inductively coupled plasma mass spectroscopy analyses, and Jeannette Woodward for their execution.
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