Cyclodipeptides: From Their Green Synthesis to Anti-Age Activity

Cyclodipeptides (CDPs) or 2,5-diketopiperazines (DKPs) are naturally occurring biomolecules that have increasingly attracted researchers’ attention for a variety of reasons, as evident from the many recent reviews that cover aspects ranging from their sustainable production via enzymatic catalysis to all the possible methods for their preparation and self-assembly into gels and their biological activities, including their function as anti-cancer agents. First of all, compared to linear dipeptide analogues, they lack the charged termini, thus being more lipophilic with a fast membrane absorption in the digestive tract, thanks to their high permeability. Secondly, they have less conformational freedom and a unique structural rigidity, which confers them with a higher resistance against enzymatic degradation, especially for those containing proline. The presence of the six-membered piperazine ring with sidechains that are oriented in a spatially defined manner allows the accurate prediction of their conformation too. Thanks to their rigid backbone, they can mimic secondary structural motifs, especially β-turns, which are present in many bioactive compounds. The spectrum of their biological activities indeed encompasses antimicrobial, antitumoral, and neuroprotective effects.

CDPs & Brain

Cyclic dipeptides (CDPs) are a group of hormone-like molecules that are evolutionarily conserved from bacteria to humans. In bacteria, CDPs are used in quorum sensing (QS) to communicate information about population size and to regulate a behavioural switch from symbiosis with their host to virulence. In mammals, CDPs have been shown to act on glial cells (macrophage-like cells) to control a conceptually homologous behavioural switch between homeostatic and inflammatory modes, with implications for the control of neurodegenerative disease.

Interestingly, DKP5 is known to be endogenous to blood, cerebrospinal fluid, brain, spinal cord, semen, and gastrointestinal tract of humans. Part of it derives from the enzymatic degradation of the thyrotropin-releasing hormone, while the origin of the rest remains to be elucidated. Interestingly, its administration reduces food intake. This DKP also has other pharmacological activities, such as regulation of body temperature, inhibition of prolactin secretion, and modulation of motor functions, which may be exerted by affecting central amine transport mechanisms. Indeed, this DKP has the remarkable ability to pass the blood–brain barrier by passive diffusion, and it has been widely studied for its ability to exert a protective role in neurodegenerative and metabolic diseases. Given its high potential for therapeutic use by oral administration also, it has been proposed as an important innovative tool to counteract neuroinflammation-based degenerative diseases.

CDPs & anti-age

Advanced glycation end products (AGEs) accumulation in the skin is known to have negative effects from an aesthetic point of view, such as the formation of wrinkles, yellowish complexion, and brown spots.

In addition, the naturally occurring, non-enzymatic glycation of proteins has been linked to several age-related pathologies. For instance, this post-translational modification was shown to affect amyloid formation and was associated with diabetes, non-alcoholic fatty liver disease, thyroid cancer progression, coronary artery disease, and pathologies of the kidneys. Therefore, the ability of DKPs to reduce AGEs could potentially have beneficial effects on health, beyond those on skin aesthetics that could be of interest for cosmetic applications.

In particular, 15 CDPs were recently tested in vitro for their anti-age properties, with the most active being cyclo(Leu-Leu), cyclo(Met-Met), and cyclo(Pro-Pro). In this work, we thus decided to test two of these as reference compounds, in addition with three more CDPs, i.e., cyclo(His-His), cyclo(His-Pro), and cyclo(His-Met). We reasoned that histidine ability to coordinate metals could serve as a useful addition, for instance to reduce the occurrence of Fenton reactions that occur during oxidative stress in aging human skin.

Skin Absorption of DKPs

The human skin is composed of three layers, which are the epidermis, the dermis, and the hypodermis. The epidermis is avascular, stratified, squamous epithelium, and its outer layer is the stratum corneum (SC). The dermis is mainly composed of fibroblasts in an extracellular matrix of proteins. Finally, the hypodermis is composed mainly of subcutaneous fat with blood and lymphatic vessels. Ideally, cosmetics should surpass the barrier composed of the SC, and reach the dermis for maximal efficacy, without passing over and potentially leading to systemic effects.

The majority of substances penetrate the skin through passive diffusion, which can be described most simply by Fick’s diffusion law. As a general rule, molecules that more readily permeate the skin have a molecular weight (MW) under 5 kDa and an octanol-water partition coefficient logP = 1–3. 

Skin absorption is a process that describes the passage of compounds across the skin and includes penetration and permeation. Dermal penetration is the mass of the test substance that enters the skin, and dermal permeation is the mass that has transferred from the skin to the reservoir compartment fluid. It is also well known that a hydrophilic substance cannot penetrate the skin easily because it cannot enter the hydrophobic SC layer, while a hydrophobic substance easily enters the SC, but it remains stored inside it since the next layer is hydrophilic.The other DKPs 1–4 all displayed higher permeation rates and the ability to surpass the SC and reach the deeper layers of the skin, i.e., the epidermis and dermis. Clearly, formulation design will be key in the future to modulate their penetration rate in the skin, as needed for the intended application.