Mechanistic Plausibility Should not be Conflated With Clinical Validation of Glycyl- L- Histidyl- L- Lysine-Copper

Mokhtar et al provide a timely synthesis of the expanding literature surrounding glycyl-L-histidyl-L-lysine-copper (GHK-Cu).1 We agree with their central conclusion that a substantial translational gap separates experimental promise from evidence-based clinical use. The review already acknowledges the predominance of preclinical evidence, heterogeneity of models and formulations, and lack of robust clinical trials. Our aim is not to restate those limitations but to identify specific residual statements and visual elements that may still convey greater clinical certainty than the evidence permits. These include the review's abstract presentation of positive clinical findings without qualification by a minimally important difference, Figures 2-4 and 7, depicting receptor binding and downstream pathways as established, and the characterization of safety as favorable based on short-duration topical studies.1 Of the 20 included studies, 18 were preclinical and only 2 were randomized controlled trials. Across these 2 trials, only 52 participants completed follow-up: 13 in Miller et al and 39 in Badenhorst et al. No significant between-group differences were demonstrated in postoperative erythema, wrinkle improvement, or overall skin appearance after carbon dioxide laser resurfacing. The only positive outcome was greater patient-reported satisfaction.2 Conversely, Badenhorst et al reported reductions in wrinkle depth and volume using a proprietary lipid-nanocarrier formulation.3 These findings are formulation-specific and cannot establish that comparable effects would occur with unencapsulated GHK-Cu, alternative topical vehicles, or injectable preparations. Moreover, the clinical importance of the measured changes was not established using a predefined minimally important difference. Although the Discussion acknowledges these limitations, the review's abstract presents the positive clinical findings with insufficient qualification. The mechanistic evidence also requires more careful causal interpretation. Copper (II) chloride (CuCl2) reproduced the stimulation of pro-matrix metalloproteinase-2 (proMMP-2) attributed to GHK-Cu, whereas free GHK and CuCl2 reproduced the suppression of transforming growth factor-β1 (TGF-β1) and interleukin-6in fibroblast experiments. These observations do not prove that GHK-Cu acts merely through nonspecific copper-mediated stress, nor do they exclude a receptor-mediated or complex-dependent contribution. They do, however, show that the intact complex is not necessary for these selected endpoints; therefore, these results alone do not establish a unique GHK-Cu signaling pathway. Conversely, other collagen and extracellular-matrix findings were not reproduced by copper alone. The available results consequently support endpoint-specific and context-dependent activity rather than a single unified mechanism. This uncertainty is not adequately conveyed in Figures 2-4 and 7. These schematics depict GHK-Cu binding to unspecified cell-surface receptors and initiating defined downstream pathways. However, the principal fibroblast studies measured downstream products such as cytokine secretion, collagen production, matrix metalloproteinases (MMP) expression, or tissue inhibitor of metalloproteinases expression. They did not identify a GHK-Cu receptor or demonstrate receptor-dependent signal transduction. In particular, the Gruchlik et al’s studies did not directly measure IκB complex phosphorylation, nuclear factor kappa B nuclear translocation, or suppressor of mothers against decapentaplegic 2/3 (SMAD2/3) activity.4 Presenting these intermediate steps as established causal events exceeds the experimental resolution of the cited evidence. These are in vitro findings from fibroblast cultures and should be treated as hypothesis-generating rather than direct evidence of receptor-dependent signaling in patient tissue. The description of a “favorable safety profile” also warrants qualification. The human evidence is limited to topical use in 2 small trials lasting only 8 and 12 weeks, involving women aged 40 to 77 years. These studies cannot establish long-term safety, safety across broader populations, or safety of injectable administration. Absence of detected toxicity in short-duration cell, animal, and topical studies should be described as preliminary tolerability rather than a clinically established safety profile. To clarify where the review's evidence supports only qualified conclusions, Tables 1 and 2 separate directly observed findings from limits of inference and indicate whether each row reflects the review's presentation, the primary studies, or our reinterpretation. Mokhtar et al have performed a useful service by organizing a scattered and biologically interesting literature. The review is methodologically structured and appropriately identifies substantial heterogeneity, but its evidentiary strength remains inseparable from that of its primary studies. Although the review included 20 studies, 18 were preclinical; the remaining clinical evidence consisted of only 2 randomized trials, with 52 participants completing follow-up in total (13 in Miller et al and 39 in Badenhorst et al). A systematic search cannot convert these predominantly preclinical, mechanistically indirect, and formulation-dependent observations into clinical validation. The review's principal contribution is therefore to define the research gap rather than to establish efficacy or safety. We do not contend that Mokhtar et al claim clinical validation. Rather, we argue that the review's evidence map may be read as more clinically affirmative unless the abstract, figures, and safety language are qualified. This letter adds an endpoint-specific analysis of mechanistic specificity, a table-based separation of directly observed evidence from limits of inference, and a qualification of safety by duration, population, and route of administration. GHK-Cu should remain framed as a plausible investigational agent and not an established regenerative treatment, until adequately powered trials demonstrate objective, clinically meaningful, and durable benefit. That distinction is essential when biological promise is already moving faster than clinical proof. Mechanistic Claims: Directly Observed Evidence and Limits of Inference Rows summarize the present authors’ interpretation. “Directly observed evidence” refers to findings reported in the cited primary studies; “Supported interpretation” reflects the authors’ qualified reading and does not imply that the original review claimed clinical validation. GHK-Cu, glycyl-L-histidyl-L-lysine-copper; MMP, matrix metalloproteinase; TIMP, tissue inhibitor of metalloproteinase; TGF, transforming growth factor; IL, interleukin; NF-κB, nuclear factor kappa B; SMAD, suppressor of mothers against decapentaplegic. Clinical Evidence: Review Summary and Limits on Certainty Rows summarize the present authors’ interpretation. “Evidence summarized in the review” reflects the review's presentation; “Implication for interpretation” reflects the authors’ qualified reading and does not imply that the original review claimed clinical validation. GHK-Cu, glycyl-L-histidyl-L-lysine-copper. The authors declared no potential conflicts of interest with respect to the research, authorship, and publication of this article. The authors received no financial support for the research, authorship, and publication of this article.